Methods and means of increasing the water use efficiency of plants

ABSTRACT

The invention relates to methods of producing a desired phenotype in a plant by manipulation of gene expression within the plant. The method relates to means which inhibit the level of PK220 gene expression or activity, wherein a desired phenotype such as increased water use efficiency relative to a wild type control plant. The invention also relates to nucleic acid sequences and constructs useful such methods and methods of generating and isolating plants having decreased PK220 expression or activity.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/483,660, filed on Jun. 12, 2009 (now allowed), which claims thebenefit of U.S. Ser. No. 61/132,067, filed Jun. 13, 2008, the contentsof each of which are incorporated herein by reference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “PREP-017_C01US SEQ LISTING.txt”,which was created on Aug. 30, 2016 and is 225 KB in size, are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention is in the field of plant molecular biology and relates totransgenic plants having novel phenotypes, methods of producing suchplants and polynucleotides and polypeptides useful in such methods. Morespecifically, the invention relates to inhibition of a protein kinaseand transgenic plants having inhibited protein kinase activity.

BACKGROUND OF THE INVENTION

Water is essential for plant survival, growth and reproduction.Assimilation of carbon dioxide by photosynthesis is directly linked towater loss through the stomata. Crop productivity which is closelylinked to biomass production is dependent on plant water use efficiency(WUE) especially in water limited conditions (Passioura 1994 andSinclair 1994, in Physiology and Determination of Crop Yield). Water useefficiency over a period of plant's growth can be calculated as theratio of biomass produced per unit of water transpired (Sinclair 1994).Instantaneous measurements of water use efficiency can also be obtainedas the ratio of carbon dioxide assimilation to transpiration using gasexchange measurements (Farquhar and Sharkey 1994, in Physiology andDetermination of Crop Yield). Since there is a close correlation betweencrop productivity and water use efficiency, many attempts have been madeto study and understand this relationship and the genetic componentsinvolved. To maximize the productivity and yield of a crop, efforts havebeen made to try to improve the water use efficiency of plants (Condonet al., 2002, Araus et al., 2002, Davies et al., 2002). Higher water useefficiency can be achieved either by increasing the biomass productionand carbon dioxide assimilation or by reducing the transpiration waterloss. Reduced transpiration, especially under non-limiting waterconditions can be associated with reduced growth rate and thereforereduced crop productivity. This poses a dilemma on how to improve cropproductivity and yield under water limited conditions but also maintainit under irrigated or non-limited water conditions (Condon et al.,2002).

Improvements to water use efficiency, to date, have used plant breedingmethods whereby high water use efficiency varieties were crossed withthe more productive but lower water use efficiency varieties in hope ofimprovements in crop yield under water limited conditions (Condon etal., 2002, Araus et al., 2002). Quantitative trait loci (QTL) approachesto identifying the components of water use efficiency have been the mostcommon methods historically used (Mian et al., 1996, Martin et al.,1989, Thumma et al., 2001, Price et al., 2002), and more recentlyattempts have been made to engineer improved plants by molecular geneticmeans.

The first gene associated with water use efficiency was ERECTA. TheERECTA gene was first identified as a gene functioning in inflorescencedevelopment and organ morphogenesis (Torii et al., 1996),). It was laterfound by QTL mapping to be a major contributor to transpirationefficiency, defined as water transpired per carbon dioxide assimilated,an opposite indicator to water use efficiency in Arabidopsis (Masle etal., 2005). ERECTA encodes a putative leucin-rich repeat receptor-likekinase (LRR-RLK). The regulatory mechanism of LRR-RLK is yet to beunderstood although it was suggested due to, at least in part, theeffects on stomatal density, epidermal cell expansion, mesophyll cellproliferation and cell-cell contact. The normal transpiration efficiencywas restored upon complementation using wild type ERECTA in mutantexacta. However, it is not known whether overexpression of ERECTA intransgenic Arabidopsis will result in reduced transpiration efficiencyor enhanced water use efficiency. It is the only report showing a plantreceptor-like kinase to be involved in transpiration efficiency or wateruse efficiency.

Another Arabidopsis gene implicated in water use efficiency is the HARDYgene, found through the phenotypic screening of an activation taggedmutant collection (Karaba et al., 2007). Overexpression of HARDY in riceresulted in improved water use efficiency by enhancing photosyntheticassimilation and reducing transpiration. The transgenic rice withincreased expression of HARDY exhibited increased shoot biomass underoptimal water conditions and increased root biomass under water limitedconditions. Overexpression of HARDY in Arabidopsis resulted in thickerleaves with more mesophyll cells and in rice increased leaf biomass andbundle sheet cells. These modifications contributed to enhancedphotosynthetic activity and efficiency (Karaba et al., 2007).

Protein kinases are a large family of enzymes that modify proteins byaddition of phosphate groups (phosphorylation). Protein kinasesconstitute about 2% of all eukaryotic genes, many of which mediate theresponse of eukaryotic cells to external stimuli. All single subunitprotein kinases contain a common catalytic domain near the carboxylterminus while the amino terminus plays a regulatory role

Plant receptor-like kinases are serine/threonine protein kinases with apredicted signal peptide at the amino terminus, a single transmembraneregion and a cytoplasmic kinase domain. There are more than 610 RLKspotentially encoded in Arabidopsis (Shiu and Bleecker 2001).Receptor-like kinases are often part of a signaling cascade. Theyinterpret extracellular signals, through ligand binding, andphosphorylate targets in a signaling cascade which in turn affectdownstream cell processes, such as gene expression (Hardie 1999).

Identification of genes that can be manipulated to provide beneficialcharacteristics is highly desirable. So too are means and methods ofutilizing the identified genes to effect the desirable characteristics.The receptor-like kinase identified as At2g25220 in the TAIR database isone serine/threonine kinase, and a member of the large gene family ofreceptor-like kinases with over 600 members in Arabidopsis (Shiu et al.,2001). However, except for annotation of the sequence as a kinase nofunction or role for the At2g25220 gene has been disclosed. In thepresent invention a high water use efficiency gene (HWE) has beenidentified that when its expression or activity is inhibited results inbeneficial phenotypes, such as, enhancement of plant biomassaccumulation relative to the water used. This occurs under both waterlimited and non-limited conditions and ensures better growth andtherefore greater productivity of the plants.

SUMMARY OF THE INVENTION

This invention is bases upon the discovery of a mutation in the PK220gene that results in a plant with an altered phenotype such for example,increased water use efficiency, increased drought tolerance, reducedsensitivity to cold temperature and reduced inhibition of seedlinggrowth in low nitrogen conditions compared to plants without themutation.

More specifically, the invention relates to the identification of amutant plant that comprises a mutation in the PK220 gene also referredto herein as the HWE gene. The PK220 gene is a receptor-like proteinkinase. Inhibition of the expression or activity of the PK220 gene inplants provides beneficial phenotypes such as improved water useefficiency in a plant. The improved water use efficiency phenotyperesults in plants having improved drought tolerance.

In one aspect the invention provides a method of producing a transgenicplant, by transforming a plant, a plant tissue culture, or a plant cellwith a vector containing a nucleic acid construct that inhibits theexpression or activity of a PK220 gene to obtain a plant, tissue cultureor a plant cell with decreased PK220 expression or activity and growingthe plant or regenerating a plant from the plant tissue culture or plantcell. wherein a plant having increased water use efficiency is produced.

Accordingly, the present invention provides a method of producing aplant having an improved property, wherein the method includesinhibiting the expression or activity of an endogenous PK220 gene,wherein a plant is produced having an advantageous phenotype or improvedproperty. In a particular embodiment, the present invention provides amethod for producing plants having increased water use efficiency,wherein the method includes include generation of transgenic plants andmodification of plants genome using the methods described herein.

Water use efficiency refers to the ratio between the amounts of biomassproduced per unit water transpired when measured gravimetrically and theratio of photosynthetic rate to the rate of transpiration when measuredusing gas exchange quantification of a leaf or shoot. As used herein,the term “increased water use efficiency” refers to a plant water useefficiency that is 2, 4, 5, 6, 8, 10, 20 or more fold greater ascompared to the water use efficiency of a corresponding wild-type plant.For example, a plant having increased water use efficiency as comparedto a wild-type plant may have 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%70%, 75% or greater water use efficiency than the correspondingwild-type plant.

The methods of the invention involve inhibiting or reduced theexpression or activity of an endogenous gene, such as PK220, wherein aplant is produced having an advantageous phenotype or improved property,such as increased water use efficiency. In one aspect, the inventionprovides a method of producing a plant having increased water useefficiency relative to a wild-type plant, by introducing into a plantcell a nucleic acid construct that inhibits or reduces the expression oractivity of PK220. For example, a plant having increased water useefficiency relative to a wild type plant is produced by a) providing anucleic acid construct containing a promoter operably linked to anucleic acid construct that inhibits PK220 activity; b) inserting thenucleic construct into a vector; c) transforming a plant, tissueculture, or a plant cell with the vector to obtain a plant, tissueculture or a plant cell with decreased PK220 activity; d) growing theplant or regenerating a plant from the tissue culture or plant cell,wherein a plant having increased water use efficiency relative to a wildtype plant is produced. The construct includes a promoter such as aconstitutive promoter, a tissue specific promoter or an induciblepromoter. Preferably, the tissue specific promoter is a root promoter. Apreferable inducible promoter is a drought inducible promoter.

The term “nucleic acid construct” refers to a full length gene sequenceor portion thereof, wherein a portion is preferably at least 19, 20, 21,22, 23, 24, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, or 150 nucleotidesin length, or the compliment thereof. Alternatively it may be anoligonucleotide, single or double stranded and made up of DNA or RNA ora DNA-RNA duplex. In a particular embodiment, the nucleic acid constructcontains the full length PK220 gene sequence, or a portion thereof,wherein the portion of the PK220 sequence is at least 19, 20, 21, 22,23, 24, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, or 150 nucleotides inlength, or its compliment.

Also provided by the invention is a transgenic plant having anadvantageous phenotype or improved property such as increased water useefficiency, produced by the methods described herein.

In another aspect the invention provides a plant having a non-naturallyoccurring mutation in an PK220 gene, wherein the plant has decreasedPK220 expression or activity and the plant has increased water useefficiency relative to a wild-type control. Decreased PK220 expressionor activity refers to a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50, 60, or 75-fold reduction or greater, at the DNA, RNA or proteinlevel of an PK220 gene as compared to wild-type PK220, or a 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 or 75 fold reduction of PK220activity as compared to wild-type PK220 activity. PK220 activityincludes but is not limited kinase activity at serine and or threonineamino acid residues of substrate polypeptides, where it participates inphosphorylation reactions.

The invention further provides a transgenic seed produced by thetransgenic plant(s) of the invention, wherein the seed produces planthaving an advantageous phenotype or improved property such as forexample, increased water use efficiency relative to a wild-type plant.

In another embodiment, the invention provides nucleic acids forexpression of nucleic acids in a plant cell to produce a transgenicplant having an advantageous phenotype or improved property such asincreased water use efficiency.

Exemplary sequences encoding a wild type PK220 gene or portion thereofthat find use in aspects of the present invention are described in SEQID NO's: 1, 7, 9, 11, 12, 13, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 84, 86, 88, 90, 92, 94, 96, 98, 100, 153, 161 and 193.Exemplary sequences encoding a mutated PK220 gene are described in SEQID NO's:3 and 5. Exemplary sequences that are useful for constructs todownregulate PK220 expression or activity are described in SEQ ID NO's:12, 13, 147, 149, 153, 161, 168 and 174. The invention further providescompositions which contain the nucleic acids of the invention forexpression in a plant cell to produce the transgenic plants describedherein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

For convenience, before further description of the present invention,certain terms employed in the specification, examples and claims aredefined herein. These definitions should be read in light of theremainder of the disclosure and as understood by a person of ordinaryskill in the art.

A “promoter sequence”, or “promoter”, means a nucleic acid sequencecapable of inducing transcription of an operably linked gene sequence ina plant cell. Promoters include for example (but not limited to)constitutive promoters, tissue specific promoters such as a rootpromoter, an inducible promoters such as a drought inducible promoter oran endogenous promoters such as a promoter normally associated with agene of interest, i.e. a PK220 gene

The term “expression cassette” means a vector construct wherein a geneor nucleic acid sequence is transcribed. Additionally, the expressedmRNA may be translated into a polypeptide.

The terms “expression” or “overexpression” are used interchangeably andmean the expression of a gene such that the transgene is expressed. Thetotal level of expression in a cell may be elevated relative to awild-type cell.

The term “non-naturally occurring mutation” refers to any method thatintroduces mutations into a plant or plant population. For example,chemical mutagenesis such as ethane methyl sulfonate or methanesulfonicacid ethyl ester, fast neutron mutagenesis, DNA insertional means suchas a T-DNA insertion or site directed mutagenesis methods.

The term “drought stress” refers to a condition where plant growth orproductivity is inhibited relative to a plant where water is notlimiting. The term “water-stress” is used synonymously andinterchangeably with the drought water stress.

The term “drought tolerance” refers to the ability of a plant tooutperform a wildtype plant under drought stress conditions or waterlimited conditions or to use less water during grow and developmentrelative to a wildtype plant.

The “term water use efficiency” is an expression of the ratio betweenthe amounts of biomass produced per unit water transpired when measuredgravimetrically and the ratio of photosynthetic rate to the rate oftranspiration when measured using gas exchange quantification of a leafor shoot.

The term “dry weight” means plant tissue that has been dried to removethe majority of the cellular water and is used synonymously andinterchangeably with the term biomass.

The term “null” is defined as a segregated sibling of a transgenic linethat has lost the inserted transgene and is therefore used a controlline.

A number of various standard abbreviations have been used throughout thedisclosure, such as g, gram; WT, wild-type; DW, dry weight; WUE, wateruse efficiency; d, day.

The term “hwe116” means a plant having a mutation in a PK220 gene.

The HWE gene is referred to as a PK220 gene sequence and a proteinencoded by a PK220 gene is referred to as a PK220 polypeptide orprotein. The terms HWE and PK220 are synonymous.

The term “PK220 nucleic acid” refers to at least a portion of a PK220nucleic acid. Similarly the term “PK220 protein” or “PK220 polypeptide”refers to at least a portion thereof. A portion is of at least 21nucleotides in length with respect to a nucleic acid and a portion of aprotein or polypeptide is at least 7 amino acids. The term “AtPK220”refers to an Arabidopsis thaliana PK220 gene, the term “BnPK220” refersto a Brassica napus PK220 gene.

The invention is based in part on the discovery of plants having animproved agronomic property, for example, increased water useefficiency, increased drought tolerance, reduced sensitivity to coldtemperature and reduced inhibition of seedling growth in low nitrogenconditions relative to a wild type control. The gene responsible for thebeneficial phenotype has been determined and shown to be an inhibitedPK220 gene.

Methods of producing a plant, including a mutant plant, a transgenicplant or genetically modified plant, having increased water useefficiency are disclosed herein. Specifically the invention identifies aPK220 gene that when expression or activity of the PK220 gene isinhibited, a plant having a beneficial phenotype is obtained.

Determining Homology Between Two or More Sequences

To determine the percent homology between two amino acid sequences orbetween two nucleic acids, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in either of thesequences being compared for optimal alignment between the sequences).The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are homologous at that position (i.e., as used hereinamino acid or nucleic acid “homology” is equivalent to amino acid ornucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch (1970). Using GCG GAPsoftware with the following settings for nucleic acid sequencecomparison: GAP creation penalty of 5.0 and GAP extension penalty of0.3, the coding region of the analogous nucleic acid sequences referredto above exhibits a degree of identity preferably of at least 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99%, with the coding sequence portion of theDNA sequence shown in SEQ ID NO: 1.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues.

Inhibition of Endogenous PK220 Expression and Activity

An aspect of the invention pertains to means and methods of inhibitingor reducing PK220 gene expression and activity, optionally, resulting inan inhibition or reduction of PK220 protein expression and activity. Theterm “PK220 expression or activity” embraces both these levels ofinhibition or reduction. Decreased PK220 expression or activity refersto a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, or 75-foldreduction or greater, at the DNA, RNA or protein level of an PK220 geneas compared to wild-type PK220, or a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60 or 75 fold reduction of PK220 protein activity ascompared to wild-type PK220 activity. PK220 protein activity includesbut is not limited kinase activity at serine and or threonine amino acidresidues of substrate polypeptides, where it participates inphosphorylation reactions. Methods of measuring serine/threonine kinaseactivity are known to those in the art.

There are numerous methods known to those skilled in the art ofachieving such inhibition that effect a variety of steps in a geneexpression pathway, for example transcriptional regulation, posttranscriptional and translational regulation. Such methods include, butare not limited to, antisense methods, RNAi constructs, including allhairpin constructs and RNAi constructs useful for inhibition bydsRNA-directed DNA methylation or inhibition by mRNA degradation orinhibition of translation, microRNA (miRNA), including artificial miRNA(amiRNA) (Schwab et al., 2006) technologies, mutagenesis and TILLINGmethods, in vivo site specific mutagenesis techniques anddominant/negative inhibition approaches.

A preferred method of gene inhibition involves RNA inhibition (RNAi)also known as hairpin constructs. A portion of the gene to inhibit isused and cloned in a sense and antisense direction having a spacerseparating the sense and antisense portions. The size of the geneportions should be at least 20 nucleotides in length and the spacer maybe a little as 13 nucleotides (Kennerdell and Carthew, 2000) in lengthand may be an intron sequence, a coding or non-coding sequence.

Antisense is a common approach wherein the target gene, or a portionthereof, is expressed in an antisense orientation resulting ininhibition of the endogenous gene expression and activity. The antisenseportions need not be a full length gene nor be 100% identical. Providedthat the antisense is at least about 70% or more identical to theendogenous target gene and of least 19, 20, 21, 22, 23, 24, 25, 30, 40,50, 60, 70, 75, 80, 90, 100, or 150 nucleotides in length. Preferably,50 nucleotides or greater in length the desired inhibition will beobtained.

Sequences encoding a wild type PK220 gene or portion thereof that areuseful in preparing constructs for PK220 inhibition include for example,SEQ ID NO's: 1, 7, 9, 11, 12, 13, 24, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 84, 86, 88, 90, 92, 94, 96, 98, 100, 153, 161 and 193.Exemplary sequences that are useful for constructs to downregulate PK220expression or activity are described in SEQ ID NO's: 12, 13, 147, 149,153, 161, 168 and 174.

When using an antisense strategy of down-regulation, inhibition ofendogenous gene activity can be selectively targeted to the gene orgenes of choice by proper selection of a fragment or portion forantisense expression. Selection of a sequence that is present in thetarget gene sequence and not present in related genes (non-target gene)or is less than 70% conserved in the non-target sequences results inspecificity of gene inhibition.

Alternatively, amiRNA inhibition can be used to inhibit gene expressionand activity in a more specific manner than other RNAi methods. Incontrast to siRNA that requires a perfect match between the small RNAand the target mRNA, amiRNA allows up to 5 mismatches with no more than2 consecutive mismatches. The construction of amiRNA needs to meetcertain criteria described in Schawab et al. (2006). This provides amethod to down-regulate a target gene expression or activity using agene portion comprising of at least a 21 nucleotide sequence of PK220.

Dominant/negative inhibition is analogous to competitive inhibition ofbiochemical reactions. Expression of a modified or mutant polypeptidethat lacks full functionality competes with the wild type or endogenouspolypeptide thereby reducing the total gene/protein activity. Forexample an expressed protein may bind to a protein complex or enzymesubunit to produce a non-functional complex. Alternatively the expressedprotein may bind substrate but not have activity to perform the nativefunction. Expression of sufficient levels of non active protein willreduce or inhibit the overall function.

Expression of PK220 genes that produce a PK220 protein that is deficientin activity can be used for dominant/negative down-regulation of geneactivity. This is analogous to competitive inhibition. A PK220polypeptide is produced that, for example, may associate with or bind toa target molecule but lacks endogenous activity. An example of such aninactive PK220 is the AtPK220 sequence isolated from the hwe116 mutantand disclosed as SEQ ID NO:3. A target molecule may be an interactingprotein of a nucleic acid sequence. In this manner the endogenous PK220protein is effectively diluted and downstream responses will beattenuated.

In vivo site specific mutagenesis is available whereby one can introducea mutation into a cells genome to create a specific mutation. The methodas essentially described in Dong et al. (2006) or US patent applicationpublication number 20060162024 which refer to the methods ofoligonucleotide-directed gene repair. Alternatively one may use chimericRNA/DNA oligonucleotides essentially as described Beetham (1999).Accordingly, a premature stop codon may be generated in the cells'endogenous gene thereby producing a specific null mutant. Alternatively,the mutation may interfere with splicing of the initial transcriptthereby creating a non-translatable mRNA or a mRNA that produces analtered polypeptide which does not possess endogenous activity.Preferable mutations that result loss or reduction of PK220 expressionor activity include a C to T conversion at nucleotide position 874 whennumbered in accordance with SEQ ID NOs: 1 or 3 or a nucleotide mutationthat results in an amino acid change from a Leucine (L) codon (CTT) to aPhenylalanine (F) codon (TTT) at amino acid position 292 when numberedin accordance with SEQ ID NOs: 2 or 4.

TILLING is a method of isolating mutations in a known gene from anEMS-mutagenized population. The population is screened by methodsessentially as described in (Greene et al., 2003).

Other strategies of gene inhibition will be apparent to the skilledworker including those not discussed here and those developed in thefuture.

Identification of AtPK220 homologues

Homologues of Arabidopsis thaliana PK220 (AtPK220) were identified usingdatabase sequence search tools, such as the Basic Local Alignment SearchTool (BLAST) (Altschul et al., 1990 and Altschul et al., 1997). Thetblastn or blastn sequence analysis programs were employed using theBLOSUM-62 scoring matrix (Henikoff and Henikoff, 1992). The output of aBLAST report provides a score that takes into account the alignment ofsimilar or identical residues and any gaps needed in order to align thesequences. The scoring matrix assigns a score for aligning any possiblepair of sequences. The P values reflect how many times one expects tosee a score occur by chance. Higher scores are preferred and a lowthreshold P value threshold is preferred. These are the sequenceidentity criteria. The tblastn sequence analysis program was used toquery a polypeptide sequence against six-way translations of sequencesin a nucleotide database. Hits with a P value less than −25, preferablyless than −70, and more preferably less than −100, were identified ashomologous sequences (exemplary selected sequence criteria). The blastnsequence analysis program was used to query a nucleotide sequenceagainst a nucleotide sequence database. In this case too, higher scoreswere preferred and a preferred threshold P value was less than −13,preferably less than −50, and more preferably less than −100.

A PK220 gene can be isolated via standard PCR amplification techniques.Use of primers to conserved regions of a PK220 gene and PCRamplification produces a fragment or full length copy of the desiredgene. Template may be DNA, genomic or a cDNA library, or RNA or mRNA foruse with reverse transcriptase PCR (RtPCR) techniques. Conserved regionscan be identified using sequence comparison tools such as BLAST orCLUSTALW for example. Suitable primers have been used and describedelsewhere in this application.

Alternatively, a fragment of a sequence from a PK220 gene is³²P-radiolabeled by random priming (Sambrook et al., 1989) and used toscreen a plant genomic library (the exemplary test polynucleotides). Asan example, total plant DNA from Arabidopsis thaliana, Nicotianatabacum, Lycopersicon pimpinellifolium, Prunus avium, Prunus cerasus,Cucumis sativus, or Oryza sativa are isolated according to Stockinger etal. (Stockinger et al., 1996). Approximately 2 to 10 μg of each DNAsample are restriction digested, transferred to nylon membrane (MicronSeparations, Westboro, Mass.) and hybridized. Hybridization conditionsare: 42° C. in 50% formamide, 5×SSC, 20 mM phosphate buffer1×Denhardt's, 10% dextran sulfate, and 100 μg/ml herring sperm DNA. Fourlow stringency washes at RT in 2×SSC, 0.05% sodium sarcosyl and 0.02%sodium pyrophosphate are performed prior to high stringency washes at55° C. in 0.2.times.SSC, 0.05% sodium sarcosyl and 0.01% sodiumpyrophosphate. High stringency washes are performed until no counts aredetected in the washout according to Walling et al. (Walling et al.,1988). Positive isolates are identified, purified and sequenced. Othermethods are available for hybridization, for example the ExpressHyb™hybridization solution available from Clonetech.

PK220 Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PK220 protein,a PK220 gene or genomic sequence or portions thereof and analogs orhomologs thereof. As used herein the term expression vector includesvectors which are designed to provide transcription of the nucleic acidsequence. Transcribed sequences may be designed to inhibit theendogenous expression or activity of an endogenous gene activitycorrelating to the transcribed sequence. Optionally, the transcribednucleic acid need not be translated but rather inhibits the endogenousgene expression as in antisense or hairpin down-regulation methodology.Alternatively, the transcribed nucleic acid may be translated into apolypeptide or protein product. The polypeptide may be a non-fulllength, mutant or modified variant of the endogenous protein. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication). Othervectors are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors”. In general, expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids. In the present specification, “plasmid” and “vector” can beused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors or plant transformationvectors, binary or otherwise, which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences) orinducible promoters (e.g., induced in response to abiotic factors suchas environmental conditions, heat, drought, nutrient status orphysiological status of the cell or biotic such as pathogen responsive).Examples of suitable promoters include for example constitutivepromoters, ABA inducible promoters, tissue specific promoters andabiotic or biotic inducible promoters. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired as well as timing and location ofexpression, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., PK220 proteins, mutant forms of PK220 proteins,fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PK220 genes, PK220 proteins, or portions thereof, inprokaryotic or eukaryotic cells. For example, PK220 genes or PK220proteins can be expressed in bacterial cells such as Escherichia coli,insect cells (using baculovirus expression vectors),) yeast cells, plantcells or mammalian cells. Suitable host cells are discussed further inGoeddel (1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

In one embodiment, a nucleic acid of the invention is expressed inplants cells using a plant expression vector. Examples of plantexpression vectors systems include tumor inducing (Ti) plasmid orportion thereof found in Agrobacterium, cauliflower mosaic virus (CaMV)DNA and vectors such as pBI121.

For expression in plants, the recombinant expression cassette willcontain in addition to the PK220 nucleic acids, a promoter region thatfunctions in a plant cell, a transcription initiation site (if thecoding sequence to transcribed lacks one), and optionally atranscription termination/polyadenylation sequence. Thetermination/polyadenylation region may be obtained from the same gene asthe promoter sequence or may be obtained from different genes. Uniquerestriction enzyme sites at the 5′ and 3′ ends of the cassette aretypically included to allow for easy insertion into a pre-existingvector.

Examples of suitable promoters include promoters from plant viruses suchas the 35S promoter from cauliflower mosaic virus (CaMV) (Odell et al.,1985), promoters from genes such as rice actin (McElroy et al., 1990),ubiquitin (Christensen et al., 1992; pEMU (Last et al., 1991), MAS(Velten et al., 1984), maize H3 histone (Lepetit et al., 1992); andAtanassvoa et al., 1992), the 5′- or 3′-promoter derived from T-DNA ofAgrobacterium tumefaciens, the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, therubisco promoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), asynthetic promoter, such as Rsyn7, SCP and UCP promoters,ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heatshock promoters, seed-specific promoters and other transcriptioninitiation regions from various plant genes, for example, including thevarious opine initiation regions, such as for example, octopine,mannopine, and nopaline. In some cases a promoter associated with thegene of interest (e.g. PK220) may be used to express a constructtargeting the gene of interest, for example the native AtPK220 promoter(P_(PK)). Additional regulatory elements that may be connected to aPK220 encoding nucleic acid sequence for expression in plant cellsinclude terminators, polyadenylation sequences, and nucleic acidsequences encoding signal peptides that permit localization within aplant cell or secretion of the protein from the cell. Such regulatoryelements and methods for adding or exchanging these elements with theregulatory elements of PK220 gene are known and include, but are notlimited to, 3′ termination and/or polyadenylation regions such as thoseof the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan etal., 1983); the potato proteinase inhibitor II (PINII) gene (Keil etal., 1986) and hereby incorporated by reference); and An et al. (1989);and the CaMV 19S gene (Mogen et al., 1990).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos et al., 1989) and the Nicotianaplumbaginifolia extension gene (De Loose et al., 1991), or signalpeptides which target proteins to the vacuole like the sweet potatosporamin gene (Matsuoka et al., 1991) and the barley lectin gene(Wilkins et al., 1990), or signals which cause proteins to be secretedsuch as that of PRIb (Lund et al., 1992), or those which target proteinsto the plastids such as that of rapeseed enoyl-ACP reductase (Verwoertet al., 1994) are useful in the invention.

In another embodiment, the recombinant expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particularcell type (e.g., tissue-specific regulatory elements are used to expressthe nucleic acid). Tissue-specific regulatory elements are known in theart. For example, the promoter associated with a coding sequenceidentified in the TAIR data base as At2g44790 (P₄₇₉₀) is a root specificpromoter. Especially useful in connection with the nucleic acids of thepresent invention are expression systems which are operable in plants.These include systems which are under control of a tissue-specificpromoter, as well as those which involve promoters that are operable inall plant tissues.

Organ-specific promoters are also well known. For example, the chalconesynthase-A gene (van der Meer et al., 1990) or thedihydroflavonol-4-reductase (dfr) promoter (Elomaa et al., 1998) directexpression in specific floral tissues. Also available are the patatinclass I promoter is transcriptionally activated only in the potato tuberand can be used to target gene expression in the tuber (Bevan, 1986).Another potato-specific promoter is the granule-bound starch synthase(GBSS) promoter (Visser et al., 1991).

Other organ-specific promoters appropriate for a desired target organcan be isolated using known procedures. These control sequences aregenerally associated with genes uniquely expressed in the desired organ.In a typical higher plant, each organ has thousands of mRNAs that areabsent from other organ systems (reviewed in Goldberg, 1986).

The resulting expression system or cassette is ligated into or otherwiseconstructed to be included in a recombinant vector which is appropriatefor plant transformation. The vector may also contain a selectablemarker gene by which transformed plant cells can be identified inculture. The marker gene may encode antibiotic resistance. These markersinclude resistance to G418, hygromycin, bleomycin, kanamycin, andgentamicin. Alternatively the marker gene may encode a herbicidetolerance gene that provides tolerance to glufosinate or glyphosate typeherbicides. After transforming the plant cells, those cells having thevector will be identified by their ability to grow on a mediumcontaining the particular antibiotic or herbicide. Replicationsequences, of bacterial or viral origin, are generally also included toallow the vector to be cloned in a bacterial or phage host, preferably abroad host range prokaryotic origin of replication is included. Aselectable marker for bacteria should also be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, in the case ofAgrobacterium transformations, T-DNA sequences will also be included forsubsequent transfer to plant chromosomes.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a polypeptide ofthe invention encoded in an open reading frame of a polynucleotide ofthe invention. Accordingly, the invention further provides methods forproducing a polypeptide using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding a polypeptide ofthe invention has been introduced) in a suitable medium such that thepolypeptide is produced. In another embodiment, the method furthercomprises isolating the polypeptide from the medium or the host cell.

A number of cell types may act as suitable host cell for expression of apolypeptide encoded by an open reading frame in a polynucleotide of theinvention. Plant host cells include, for example, plant cells that couldfunction as suitable hosts for the expression of a polynucleotide of theinvention include epidermal cells, mesophyll and other ground tissues,and vascular tissues in leaves, stems, floral organs, and roots from avariety of plant species, such as Arabidopsis thaliana, Nicotianatabacum, Brassica napus, Zea mays, Oryza sativa, Gossypium hirsutum andGlycine max.

Expression of PK220 nucleic acids encoding a PK220 protein that is notfully functional can be useful in a dominant/negative inhibition method.A PK220 variant polypeptide, or portion thereof, is expressed in a plantsuch that it has partial functionality. The variant polypeptide may forexample have the ability to bind other molecules but does not permitproper activity of the complex, resulting in overall inhibition of PK220activity.

Transformed Plants Cells and Transgenic Plants

The invention includes a protoplast, plants cell, plant tissue and plant(e.g., monocot or dicot) transformed with a PK220 nucleic acid, a vectorcontaining a PK220 nucleic acid or an expression vector containing aPK220 nucleic acid. As used herein, “plant” is meant to include not onlya whole plant but also a portion thereof (i.e., cells, and tissues,including for example, leaves, stems, shoots, roots, flowers, fruits andseeds).

The plant can be any plant type including, for example, species from thegenera Arabidopsis, Brassica, Oryza, Zea, Sorghum, Brachypodium,Miscanthus, Gossypium, Triticum, Glycine, Pisum, Phaseolus,Lycopersicon, Trifolium, Cannabis, Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum,Geranium, Manihot, Daucus, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Brow aalia, Lolium, Avena, Hordeum, Secale,Picea, Caco, and Populus.

The invention also includes cells, tissues, including for example,leaves, stems, shoots, roots, flowers, fruits and seeds and the progenyderived from the transformed plant.

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a gene into a plant host, including biological andphysical plant transformation protocols (See, for example, Miki et al.,(1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pages 67-88; and Andrew Bent in, Clough S Jand Bent A F, (1998) “Floral dipping: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana”). Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, polyethylene glycol(PEG) transformation, microorganism-mediated gene transfer such asAgrobacterium (Horsch et al., 1985), electroporation, protoplasttransformation, micro-injection, flower dipping and biolisticbombardment.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium tumefaciens and A. rhizogenes which are plant pathogenicbacteria which genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants (See, for example,Kado, 1991). Descriptions of the Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided in Gruberet al. (1993). and Moloney et al., (1989).

Transgenic Arabidopsis plants can be produced easily by the method ofdipping flowering plants into an Agrobacterium culture, based on themethod of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping:a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Wild type plants are grown until the plant hasboth developing flowers and open flowers. The plants are inverted for 1minute into a solution of Agrobacterium culture carrying the appropriategene construct. Plants are then left horizontal in a tray and keptcovered for two days to maintain humidity and then righted and bagged tocontinue growth and seed development. Mature seed is bulk harvested.

Direct Gene Transfer

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford etal., 1993; Klein et al., 1992).

Plant transformation can also be achieved by the Aerosol Beam Injector(ABI) method described in U.S. Pat. No. 5,240,842, U.S. Pat. No.6,809,232. Aerosol beam technology is used to accelerate wet or dryparticles to speeds enabling the particles to penetrate living cells.Aerosol beam technology employs the jet expansion of an inert gas as itpasses from a region of higher gas pressure to a region of lower gaspressure through a small orifice. The expanding gas accelerates aerosoldroplets, containing nucleic acid molecules to be introduced into a cellor tissue. The accelerated particles are positioned to impact apreferred target, for example a plant cell. The particles areconstructed as droplets of a sufficiently small size so that the cellsurvives the penetration. The transformed cell or tissue is grown toproduce a plant by standard techniques known to those in the applicableart.

Regeneration of Transformants

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

A preferred transgenic plant is an independent segregate and cantransmit the PK220 gene construct to its progeny. A more preferredtransgenic plant is homozygous for the gene construct, and transmitsthat gene construct to all offspring on sexual mating. Seed from atransgenic plant may be grown in the field or greenhouse, and resultingsexually mature transgenic plants are self-pollinated to generate truebreeding plants. The progeny from these plants become true breedinglines that are evaluated for decreased expression of the PK220 gene.

Method of Producing Transgenic Plants

Also included in the invention are methods of producing a transgenicplant having increased water use efficiency, reduced sensitivity to coldtemperature and reduced inhibition of seedling growth in low nitrogenconditions, relative to a wild type plant. The method includesintroducing into one or more plant cells a compound that inhibits orreduces PK220 expression or activity in the plant to generate atransgenic plant cell and regenerating a transgenic plant from thetransgenic cell. The compound can be, e.g., (i) a PK220 polypeptide;(ii) a PK220 nucleic acid, analog, homologue, orthologue, portion,variant or complement thereof; (iii) a nucleic acid that decreasesexpression of a PK220 nucleic acid. A nucleic acid that decreasesexpression of a PK220 nucleic acid may include promoters or enhancerelements. The PK220 nucleic acid can be either endogenous or exogenous,for example an Arabidoposis PK220 nucleic acid may be introduced into aBrassica or corn species. Preferably, the compound is a PK220 nucleicacid sequence endogenous to the species being transformed.Alternatively, the compound is a PK220 nucleic acid sequence exogenousto the species being transformed and having at least 70%, 75%, 80%, 85%,90% or greater homology to the endogenous target sequence.

In various aspects the transgenic plant has an altered phenotype ascompared to a wild type plant (i.e., untransformed). By alteredphenotype is meant that the plant has a one or more characteristic thatis different from the wild type plant. For example, when the transgenicplant has been contacted with a compound that decreases the expressionor activity of a PK220 nucleic acid, the plant has a phenotype such asincreased water use efficiency, reduced sensitivity to cold temperatureand reduced inhibition of seedling growth in low nitrogen conditions,relative to a wild type plant.

The plant can be any plant type including, for example, species from thegenera Arabidopsis, Brassica, Oryza, Zea, Sorghum, Brachypodium,Miscanthus, Gossypium, Triticum, Glycine, Pisum, PhaseolusLycopersicon,Trifolium, Cannabis, Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus,Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Browaalia, Lolium, Avena, Hordeum, Secale, Picea,Caco, and Populus.

Examples Identification of High Water Use Efficiency Mutant hwe116

An Arabidopsis EMS mutant (Columbia background) was identified initiallyas having drought tolerant properties. The mutant was tested for wateruse efficiency under optimal and drought conditions. The result showedthat the drought tolerant nature of this mutant is due to its higherwater use efficiency under both water stressed and optimal waterconditions. Thus, this mutant is named hwe116.

Map Based Cloning of hwe116

A F2 population was generated by crossing the hwe116 mutant to theLandsberg erecta (Ler) ecotype of Arabidopsis thaliana and the resultingpopulation was used for map-based cloning by assaying for droughttolerance and subsequently confirming the presence of the higher wateruse efficiency trait in the mutant. The water-loss per unit dry weightof the F2 plants was measured over a 5-day drought treatment and thedata was normalized for QTL analysis relative to the hwe116 mutant andthe two wild type ecotypes, Landsberg erecta and Columbia. Leaf tissueswere collected from all F2 and control plants used in the phenotypingexperiments for genotyping. QTL analysis was conducted using MAPMAKER3.0 and WinQTLCart 2.5. To further specify the mutations within the QTLpeak, celery endonuclease I (CEL I) was used.

Mutation Detection Using CEL I Nuclease

Celery endonuclease I (CEL I), cleaves DNA with high specificity atsites of base-pair substitution that creates a mismatch between wildtype and mutant alleles and has been reportedly used for detectingmutations in EMS mutants (Yang et al., 2000; Oleykowski et al., 1998).

DNA fragments of about 5 kb were amplified by optimized PCR using hwe116or parent Columbia genomic DNA as template. Equal amounts of theamplified products were mixed together and then subjected to a cycle ofdenaturing and annealing to form heteroduplex DNA. Incubation with CEL Iat 42° C. for 20 minutes cleaves the heteroduplex DNA at points ofmutation, and DNA fragments were visualized by 1% agarose gelelectrophoresis and ethidium bromide staining.

Using this method a 5 kb PCR product was amplified using primers SEQ IDNO:102 and SEQ ID NO:104, and templates: hwe116, and the controlColumbia type. The heteroduplexes formed PCR products resulted insmaller fragments (1.4 and 3.6 kb) after CEL I digestion. Overlappingsub-fragments (about 3 kb) were amplified using primers SEQ ID NO:104and SEQ ID NO:105 to more narrowly define the mutation location. Thesub-fragment was sequenced and a C nucleotide was found to have beenmutated to T nucleotide in hwe116.

The mutation of interest was identified as a C to T conversion atnucleotide position 874 of SEQ ID NO's:1 and 3 that resulted in an aminoacid change from a Leucine (L) codon (CTT) to a Phenylalanine (F) codon(TTT) at amino acid position 292. The gene harboring the mutation wasidentified as a Serine/Threonine protein kinase (Ser/Thr PK). The wildtype gene was identified as being identical to Genbank Accession NumberAt2g25220. This Ser/Thr protein kinase is referred to as AtPK220 herein,and the mutated form identified in hwe116 is referred to asAtPK220L292F.

Transcriptional Evaluation

Northern analysis and RT-PCR indicate that the expression level andtranscript size of the AtPK220 gene in hwe116 is unchanged relative tothe wild type control.

Initial Cloning of Partial AtPK220L292F and AtPK220 Sequences

Based on the TAIR annotation, partial sequences of AtPK220L292F(AtPK220L292F(p)) and partial AtPK220 (AtPK220(p)) were amplified byRT-PCRs using the primers SEQ ID NO:106 and SEQ ID NO:107 which includedBamHI and PstI restriction sites for cloning and template RNA isolatedfrom hwe116 and the control plant (Columbia), respectively). Theresulting partial AtPK220L292F nucleotide sequence is shown as SEQ IDNO:5 and the corresponding amino acid sequence as SEQ ID NO:6. Theresulting partial AtPK220 nucleotide sequence is shown as SEQ ID NO:7and the corresponding amino acid sequence as SEQ ID NO:8.

Kinase Activity Assay of a Partial AtPK220L292F Protein Expressed in E.coli

The PCR products were digested with BamHI and PstI, and inserted intothe expression vector: pMAL-c2 (New England Biolabs, Beverly, Mass.) toform an in-frame fusion protein with the malE gene for expression of themaltose-binding protein: MBP-AtPK220L292F(p) and MBP-AtPK220(p). Thefusion proteins were expressed in E. coli and purified usingamylose-affinity chromatography as described by the manufacturer (NewEngland Biolabs). Fractions containing the fusion proteins were pooledand concentrated (Centriprep-30 concentrator, Amicon). SDS-PAGE was usedto analyze the expression level, size and purity of the fusion proteins.

Activity assays were carried out according to (Huang et al., 2000). Thekinase autophosphorylation assay mixtures (30 μl) contained kinasereaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 10 mM MnCl₂), 1 μCi[γ-³²P] ATP and 10 ng of purified AtPK220L292F(p) or MBP-AtPK220(p). Forthe trans-phosphorylation assays, myelin basic protein (3 μg) was addedto each assay. The reactions were started by the addition of theenzymes. After incubation at room temperature for 30 min, the reactionswere terminated by the addition of 30 μl of Laemmli sample buffer(Laemmli, 1970). The samples were heated at 95° C. for 5 min and thenloaded on a 15% SDS-polyacrylamide gel. The gels were stained withCoomassie blue R-250, then de-stained and dried. The ³²P-labeled bandswere detected using Kodak X-Omat AR film.

The wild type MBP-AtPK220(p) fusion protein was able to phosphorylatethe artificial substrate in the in vitro activity assay, indicating thatthe assay system was effective and the MBP-AtPK220(p) fusion protein wascapable of activity. In contrast, the hwe116 mutant form,MBP-AtPK220L292F(p), was unable to catalyse phosphorylation of the modelsubstrate. The single point mutation is sufficient to abolish activityof the AtPK220(p) gene from hwe116.

Isolation of Full-Length cDNA Sequence of AtPK220

The annotation of AtPK220 (At2g25220) in the TAIR database identifies a5′ start codon, termination signal and 3′ UTR sequence. Analysis of the5′ portion of the annotated sequence suggested an alternative 5′sequence and start codon location. To determine the AtPK220 genes' 5′region and the likely start codon SMART RACE (Rapid Amplification ofcDNA Ends, CloneTech) was performed.

A specific primer, SEQ ID NO:108, was designed for the 5′ RACE andyielded a 450 bp PCR product. Sequence data obtained of the 450 bp 5′RACE product indicated that the TAIR annotation of AtPK220 was missingthe 5′ 186 bp that included 39 bp of 5′ UTR sequence and 147 bp ofcoding sequence. An intron of 324 bp, located 8 bp upstream of the TAIRidentified ATG start codon of AtPK220 was also missing from the genomicannotation in TAIR.

Compiling the 5′ RACE results and TAIR database annotation yields thefull-length cDNA of AtPK220 (SEQ ID NO:9). The sequence was determinedto be 1542 bp in length, which included 39 bp of 5′ UTR, 204 bp of3′UTR, and 1299 bp of coding region. The AtPK220 coding region isidentified as SEQ ID NO:1 and encodes a protein of 432 amino acids andis identified as SEQ ID NO:2. Comparison of AtPK220 to its closesthomolog, At4g32000, shows an additional sequence of 51 bp is present inAtPK220, that includes the sequence of nucleotides 368-418 of SEQ IDNO:9. This sequence provides a target sequence for down-regulationconstructs designed to specifically down-regulate the AtPK220 gene butnot non-target genes such as At4g32000.

Sequence analysis of AtPK220 indicates that this Ser/Thr PK belongs to areceptor-like protein kinase family, possessing a signal peptide (1-29),an extracellular domain (30-67), a single transmembrane domain (68-88),an ATP-binding domain (152-175 as determined by Prosite) a Ser/Thrprotein kinase active-site domain (267-279 as determined by the InterPromethod) and an activation loop (289-298, 303-316).

Rescue of the hwell6 Mutant by AtPK220

Constructs for the expression of wild-type AtPK220 were generated andtransformed into the hwe116 mutant. The construct was constitutivelyexpressed from a CaMV 35S promoter and referred to as 35S-AtPK220.

35S-AtPK220

The primer pair SEQ ID NO:109 and SEQ ID NO:110 was used to amplify afragment comprising the full length open reading frame (ORF) of AtPK220.The primer pair SEQ ID NO:111 and SEQ ID NO:110 was used to amplify afragment comprising a portion of AtPK220 ORF. The amplified fragmentswere digested with restriction enzymes SmaI and BamHI and cloned into apEGAD vector digested with the same restriction enzymes. The fragmentcomprising the full length open reading frame of AtPK220 resulting fromthe PCR and subsequent restriction digestion is disclosed as SEQ IDNO:10. The fragment comprising a portion of the AtPK220 ORF resultingfrom the PCR and subsequent restriction digestion is disclosed as SEQ IDNO:11.

The 35S-AtPK220 construct was transformed into Arabidopsis hwe116. Thetransgenic lines were recovered and advanced to T3 homozygous lines.These lines are tested for their drought tolerance and water useefficiency characteristics. The 35S-AtPK220 construct restores the wildtype phenotypes.

T-DNA Knockout Lines and Physiology Assessment

SALK T-DNA knockout lines of AtPK220 and two close homologous genes inwhich are identified as TAIR Accession numbers AT4G32000 (SEQ ID NO:16)and AT5G11020 (SEQ ID NO:18) were obtained from ABRC and advanced tohomozygosity. They are listed as follows;

AtPK220: SALK_147838;

AtPK32000 (AT4G32000): SALK_060167, SALK 029937 and SALK 121979;

AtPK11020 (AT5G11020): SAIL_1260_H05.

Analysis of gene expression levels by either RT-PCR or Northern analysisdemonstrated that the target genes in the knockout lines was eithersignificantly reduced or completely abolished. These knockout lines wereused for physiological assessment. Only the knockout line of AtPK220(SALK_147838) showed significant drought tolerance and higher water useefficiency, indicating that AtPK220 is the target gene and responsiblefor the water use efficiency phenotype of hwe116. The closely relatedgenes AT4G32000 and AT5G11020 are not functionally redundant andinhibition of these genes is insufficient to generate the hwe116phenotype.

Inhibition of the Protein Activity for PK220 in Arabidopsis

Inhibition of gene activity can be achieved by a variety of technicalmeans, for example, antisense expression, RNAi or hairpin constructs, invivo mutagenesis, dominant negative approaches or generation of a mutantpopulation and selection of appropriate lines by screening means.Provided are examples of said means to produce plants having inhibitedPK220 gene expression and or activity.

Down-Regulation of PK220 by RNAi

Constructs were designed for RNAi inhibition of PK220 using hairpin (HP)constructs. The constructs comprised a 288 bp or a 154 bp of AtPK220cDNA sequence to produce constructs referred to as (270)PK220 and(150)PK220. The 288 bp (270)PK220 fragment comprises 10 bp of intronsequence that was included in the PCR primer during construction ofthese PCR products. Vector constructs using these fragments can be madeto drive expression under the control of a promoter of choice that willbe apparent to one of skill in the art. In these examples a constitutivepromoter (35S CaMV), or the native AtPK220 promoter (P_(PK)) was used.Two fragments, or portions, of the AtPK220 gene were selected, first a288 bp fragment At(270)PK220 (SEQ ID NO:13) and second a 154 bp fragmentAt(150)PK220 (SEQ ID NO:12) were selected from a divergent region ofAtPK220 as compared to its closest homologue At4g32000.

35S-HP-At(270)PK220 and 35S-HP-At(150)PK220

The hairpin constructs (HP) 35S-HP-At(270)PK220 and 35S-HP-At(150)PK220constructs were generated as follows. The sense fragments of (270)PK220and (150)PK220 were amplified by RT-PCR using primer pairs of SEQ IDNO:134/SEQ ID NO:115 and SEQ ID NO:114/SEQ ID NO:115, respectively. ThePCR products were digested with SacI, and inserted into a binary vectorpBI121tGUS at the SacI site, respectively. The resulting vectors werethen used to subclone the antisense fragments of (270)PK220 and(150)PK220 that were derived from RT-PCR products amplified using primerpairs of SEQ ID NO:112/SEQ ID NO:117, and SEQ ID NO:116/SEQ ID NO:117,respectively. Both the vector and PCR products were digested with BamHIand XbaI for subcloning.

P_(PK)-HP-At(270)PK220 and P_(PK)-HP-At(150)PK220

The P_(PK)-HP-At(270)PK220 and P_(PK)-HP-At(150)PK220 constructs weremade from 355-HP-At(270)PK220 or 355-HP-At(150)PK220 respectively byreplacing the 35S promoter sequence with AtPK220 promoter sequence (SEQID NO:14). The 35S promoter sequence was removed from355-HP-At(270)PK220 and 355-HP-At(150)PK220 by Hind III and Xba I doubledigestion. The linearized plasmid was then treated with Klenow fragmentof DNA polymerase I to generate blunt ends and self-ligated to form anew plasmid, in which XbaI site was restored while Hind III was gone. Byusing this restored XbaI site, a Nhe I DNA fragment of AtPK220 promoterwas cloned upstream of HP-At(270) and HP-At(150) sequence to produce thefinal plasmids of P_(PK)-HP-At(270)PK220 and P_(PK)-HP-At(150)PK220.AtPK220 promoter sequence (SEQ ID NO:14) was amplified by PCR fromArabidopsis (Columbia) genome using primer pairs of SEQ ID NO:135/SEQ IDNO:136.

P₄₇₉₀-HP-At(270)PK220

To specifically down-regulate endogenous AtPK220, a strong root promoterP₄₇₉₀ was identified and found to be highly expressed in the roots ofArabidopsis, particularly in the endodermis, pericycle, and stele. TheP₄₇₉₀ promoter is associated with a coding sequence identified asAt2g44790 and the expression characteristics of P₄₇₉₀ are similar tothat of wild type AtPK220 expression. The P₄₇₉₀ was used to replace theconstitutive 35S promoter in 35S-HP-At(270)PK220. The promoter ofAt2g44790 was amplified using Arabidopsis (Col) genomic DNA as templateand primers SEQ ID NO:151 and SEQ ID NO:152. The amplified promoterfragment has the length of 1475 base-pairs right upstream the ATG startcodon of At2g44790 according to TAIR annotation. The 1475 bp-P₄₇₉₀fragment is identified a SEQ ID NO:150. Hind III and Xba I restrictionsites were introduced to the 5′ and 3′ end of the promoter fragment byprimer design. The promoter sequence was then used to replace the 35Spromoter in 355-HP-At(270)PK plasmids by HindIII/XbaI double digestion,which resulted in the final constructs of pBI-P4790-HP-At(270)PK.

Down-Regulation of BnPK220 in Brassica Using RNAi 35S-HP-Bn(340)PK

To down-regulate the AtPK220 homolog in Brassica species, a hairpinconstruct was made using a 338 bp fragment of BnPK220 (SEQ ID NO;153) asthe sense and anti-sense portions, and pBI300tGUS as the vector. Twopairs of primers SEQ ID NO:154 and SEQ ID NO:155; and SEQ ID NO:156 andSEQ ID NO:157 with unique restriction sites were designed according toBnPK220 sequence. A PCR fragment of 338 bp in length was amplified usingBrassica napus cDNA as the template and the two pairs of primers,respectively. The SacI fragment was then inserted into pBI300tGUS at theSacI site downstream of the tGUS spacer in an antisense orientation. Theresulting plasmid was subsequently used for cloning of a XbaI-BamIfragment in a sense orientation at the XbaI and BamHI sites. The vectorpBI121tGUS was modified within the NPT II selectable marker gene andnamed pBI300. The NPT II gene in the vector pBI121 contains a pointmutation (G to T at position 3383, amino acid change E182D). To restorethe gene with its WT version, the NheI-BstBI fragment (positions2715-3648) was replaced with the corresponding NheI-BstBI fragment fromplasmid pRD400 (PNAS, 87:3435-3439, 1990; Gene, 122:383-384, 1992).

P₄₇₉₀-HP-Bn(340)PK

The P₄₇₉₀ promoter of At2g44790 was used to control expression of ahairpin construct to down-regulate endogenous BnPK220 in Brassica. Theplasmid of 355-HP-Bn(340)PK was digested with HindIII and XbaI toreplace the 35S promoter with the P₄₇₉₀ promoter.

Down-Regulation of PK220 by Antisense

The construct 35S-antisenseAtPK220 was made to down-regulate expressionof AtPK220 via antisense. The antisense fragment was generated using PCRand the primer pair SEQ ID NO:106/SEQ ID NO:113. The synthesised productwas digested with BamHI and XbaI to yield a 1177 bp sequence comprising1160 bp of AtPK220 (SEQ ID NO:11). Included at the 5′ end were 10 bp ofintron sequence and at the 3′ end, 7 bp of 3′ UTR sequence, which wereretained from the PCR primers. The 1177 bp fragment was cloned in anantisense orientation to the 35S promoter in pBI121w/oGUS at the BamHIand XbaI.

Down-Regulation of PK220 by AmiRNA

An artificial microRNA (amiRNA) construct was also made to down-regulatethe expression of AtPK220 in Arabidopsis. An Arabidopsis genomic DNAfragment containing microRNA319a gene (SEQ ID NO:148), was amplified byPCR using Arabidopsis (Col) genomic DNA as template and primers listedas SEQ ID NO:141 and SEQ ID NO:142. The backbone of miR319a was thenused to construct amiRPK220 (SEQ ID NO:149), in which a 21 bp fragmentof miRNA319a gene in both antisense and sense orientations was replacedby a 21 bp DNA fragment of AtPK220 using recombinant PCR. Three pairs ofprimers: SEQ ID NO:141/SEQ ID NO:144; SEQ ID NO:143/SEQ ID NO:146 andSEQ ID NO:145/SEQ ID NO:142 were designed for the construction. Thefinal PCR product was digested with BamHI and XbaI, and subsequentlycloned into pBI121w/o GUS for transformation into Arabidopsis or otherplant species of choice.

Inhibition of PK220 Via Dominant-Negative Strategy 35S-AtPK220L292F

For expression of a non-functional AtPK220 sequence the AtPK220L292Ffrom hwe116 was PCR amplified by RT-PCR using forward and reverseprimers SEQ ID NO:118 and SEQ ID NO:110. The PCR product was digestedwith the restriction enzymes BamHI and XbaI (SEQ ID NO:121) and ligatedinto the binary vector pBI121w/o GUS. The sequence of SEQ ID NO:121comprises the AtPK220L292F open reading frame (SEQ ID NO:3) and anadditional 3 bp at the 5′ end and 7 bp at the 3′ end that are derivedfrom UTR sequences (SEQ ID NO:121). The final construct,35S-AtPK220L292F, was used to generate Arabidopsis and Brassicatransgenic plants that were advanced to homozygosity for physiologyassessment. Additionally, the vector is used to transform a plantspecies of choice and can be a dicot or a monocot.

P₄₇₉₀-AtPK220L292F

The HindIII-XbaI fragment of the root promoter P₄₇₉₀ was used to replace35S promoter in pBI300, and then AtPK220L292F sequence was putdownstream P₄₇₉₀ by XbaI and BamHI digestion to generate theP₄₇₉₀-driven dominant-negative construct. The resulting plasmid was thenused for Brassica transformation. Additionally, the vector is used totransform a plant species of choice and can be a dicot or a monocot.

Down-Regulation of AtPK220 Homologs in a Monocot Species Using RNAiP_(BdUBQ)-HP-Bd(272)PK

An expression cassette was constructed and inserted into two differentvector backbones, the first being into the PacI-AscI sites of pUCAP andthe second being into the PacI-AscI sites of pBF012. pBF012 is identicalto pBINPLUS/ARS except that the potato-Ubi3 driven NPTII cassette hasbeen excised via FseI digestion followed by self-ligation.

Brachypodium distachyon PK220 (BdPK220) was amplified using primercombinations SEQ ID NO:158 (bWET XbaI F) plus SEQ ID NO:159 (bWET BamHIR) having XbaI or BamHI sites respectively in the primers and SEQ IDNO:158 (bWET XbaI F) plus SEQ ID NO:160 (bWET ClaI R) having XbaI orClaI sites respectively in the primers. PCR products were digested withthe indicated restriction enzymes giving a 272 bp fragment (SEQ IDNO:161).

The hairpin spacer sequence, BdWx intron 1 (SEQ ID NO:164), wasamplified with SEQ ID NO:162 (bWx BamHI F) plus SEQ ID NO:163 (bWx ClaIR) primers having BamHI or ClaI sites respectively in the primers anddigested with the indicated restriction enzymes. The B. distachyon Wxgene is a homologue of the rice GBSS waxy gene, although the intronsshow little conservation.

The three fragments were ligated together into the XbaI site of thepUCAP MCS resulting in BdWx intron 1 sequence being flanked byBd(272)PK220 target sequences in opposite orientations. The B.distachyon ubiquitin (BdUBQ) promoter contains an internal BamHI site,so the RNAi cassette was amplified with primers SEQ ID NO:200 (bWETBamHI end1) and SEQ ID NO:165 (bWET BamHI end2) which create BamHIcohesive ends without the need for BamHI digestion. The BamHI RNAifragment was then ligated into the BamHI site of pUCAP alreadycontaining BdUBQ promoter and BdUBQT terminator resulting in theintermediate clone pBF067. The pBF067 complete insert was amplified withSEQ ID NO:166 (BdUBQ PvuI F) and SEQ ID NO:167 (BdUBQT Pad R), digestedwith PvuI and Pad and subsequently ligated into the Pad site of pUCAP orpBF012 vectors already containing a BdGOS2 driven mutant NPTIIselectable marker in the AscI-PacI sites, resulting in pBF108 andpBF109, respectively. This mutant NPTII gene is commonly found incloning vectors. There is only a single base pair difference from thewild type.

This cassette is in the PacI-AscI sites of pUCAP for theshuttle/bombardment vector pBF108 and in the Pac-AscI sites of pBF012for the binary vector pBF109.

P_(BdUBQ)-HP-Pv(251)PK

An expression cassette was constructed and inserted into two differentvector backbones, the first being into the PacI-AscI sites of pUCAP andthe second being into the PacI-AscI sites of pBF012. A fragment ofPanicum virgatum PK220 being 251 bp in length (Pv(251)PK220) andidentified as SEQ ID NO:168 was amplified using primer combinations SEQID NO:169 (PvWET XbaI F) plus SEQ ID NO:170 (PvWET BamHI R) and SEQ IDNO:169 (PvWET XbaI F) plus SEQ ID NO:171 (PvWET ClaI R). PCR productswere digested with the indicated restriction enzymes. No sequenceinformation exists regarding the PvWx intron 1 so the BdWx intron 1 wasused as the spacer sequence in this construct. This sequence wasamplified with SEQ ID NO:162 (bWx BamHI F) plus SEQ ID NO:163 (bWx ClaIR) primers and digested with the indicated restriction enzymes.

The three fragments were then ligated together into the XbaI site of thepUCAP MCS resulting in BdWx intron 1 sequence being flanked byPv(251)PK220 target sequences in opposite orientations. No PvUBQpromoter sequence was available so the BdUBQ promoter and terminator areused in this construct. The BdUBQ promoter contains an internal BamHIsite, so the RNAi cassette was amplified with primers SEQ ID NO:172(PvWET BamHI end1) and SEQ ID NO:173 (PvWET BamHI end2) which createBamHI cohesive ends without the need for BamHI digestion. The BamHI RNAifragment was then ligated into the BamHI site of pUCAP alreadycontaining BdUBQ promoter and BdUBQT terminator resulting in theintermediate clone pBF152. The pBF152 complete insert was amplified withSEQ ID NO:166 (BdUBQ PvuI F) and SEQ ID NO:167 (BdUBQT PacI R), digestedwith PvuI and PacI and subsequently ligated into the PacI site of pUCAPor pBF012 vectors already containing BdGOS2 driven wildtype NPTII in theAscI-PacI sites, resulting in pBF169 and pBF170, respectively.

P_(SbUBQ)-HP-Sb(261)PK

An expression cassette was constructed and inserted into two differentvector backbones, the first being into the PacI-AscI sites of pUCAP andthe second being into the PacI-AscI sites of pBF012. A fragment ofSorghum bicolor PK220 (SbPK220) being 261 bp in length (Sb(261)PK220)and identified as SEQ ID NO:174 was amplified using primer combinationsSEQ ID NO:175 (SbWET XbaI F) plus SEQ ID NO:176 (SbWET BamHI R) and SEQID NO:175 (SbWET XbaI F) plus SEQ ID NO:177 (SbWET ClaI R). PCR productswere digested with the indicated restriction enzymes to give aSb(261)PK220 fragment. The hairpin spacer sequence, SbWx intron 1 (SEQID NO:178), was amplified with primers SEQ ID NO:179 (SbWx BamHI) plusSEQ ID NO:180 (SbWx ClaI R) and digested with the indicated restrictionenzymes. The three fragments were then ligated together into the XbaIsite of the pUCAP MCS resulting in SbWx intron 1 sequence being flankedby SbWET target sequences in opposite orientations. BamHI cohesive endswere added to the RNAi cassette via amplification with primers SEQ IDNO:181 (SbWET BamHI end1) and SEQ ID NO:182 (SbWET BamHI end2). TheBamHI RNAi fragment was then ligated into the BamHI site of pUCAPalready containing SbUBQ promoter and SbUBQT terminator resulting in theintermediate clone pBF151. The pBF151 complete insert was amplified withSEQ ID NO:192 (SbUBQ PvuI F) and SEQ ID NO:167 (BdUBQT PacI R), digestedwith PvuI and PacI and subsequently ligated into the PacI site of pUCAPor pBF012 vectors already containing BdGOS2 driven wildtype NPTII in theAscI-PacI sites, resulting in pBF158 and pBF171, respectively.

A SbGOS2 promoter was identified from the Sorghum genome sequence wasamplified and using the primer pair SEQ ID NO:184 (SbGOS2 HindIII F) andSEQ ID NO:185 (SbGOS2 HindIII R) a 1000 bp fragment of the GOS2promoter, identified as SEQ ID NO:183, was PCR amplified and clonedusing the HindIII restriction sites.

A SbUBQ promoter was identified from the Sorghum genome sequence wasamplified and using the primer pair SEQ ID NO:187 (SbUBQ PstI F) and SEQID NO:188 (SbUBQ PstI R) a 1000 bp fragment of the UBQ promoter,identified as SEQ ID NO:186, was PCR amplified and cloned using the PstIrestriction sites.

A SbUBQ terminator was identified from the Sorghum genome sequence wasamplified and using the primer pair SEQ ID NO:190 (SbUBQT KpnI F) andSEQ ID NO:191 (SbUBQT KpnI R) a 239 bp fragment of the UBQ terminator,identified as SEQ ID NO:189, was PCR amplified and cloned using the KpnIrestriction sites.

Miscanthus giganteus (MgPK220) RNAi

Expression constructs designed to down regulate via a hairpin strategycan be devised following the same strategy as described above. Resultingin a construct that may comprise the following elements, aBdGOS2-wtNPTII-BdUBQT selectable marker cassette and a BdUBQ-(MgPK220hairpin-RNAi cassette)-BdUBQT in a vector of choice such as pUCAP andpBF012

AtPK220 Promoter Isolation and Cloning

The AtPK220 promoter was isolated using a PCR approach using Arabidopsis(Columbia ecotype) genomic DNA as template. The 5′ primer, SEQ IDNO:119, was designed near the adjacent gene and the 3′ primer, SEQ IDNO:120, located 25 bp upstream of the ATG start codon of the AtPK220gene. The amplified product was digested with BamHI and SmaI and clonedinto pBI101. The digested fragment, SEQ ID NO:14, was 1510 bp in length.The resulting construct was named P_(AtPK220)-GUS.

AtPK220 Promoter Activity Analysis Using GUS Assay

P_(AtPk220)-GUS was transformed into Arabidopsis plants using flowerdipping, and the transgenic plants were advanced to T3 homozygorsity.Various tissues including young seedlings and leaves, stems, flowers,siliques, and roots from T3 flowering plants were collected, stained inX-Gluc solution at 37 C overnight, de-stained with ethanol solution, andexamined under a microscope. The results showed that the promoter ofAtPK220 was expressed mainly in endodermis and pericycle cells of roottissue and was also found in leaf trichomes and seed coat of developingseeds. Of significance was the observation that expression ofP_(AtPK220)-GUS was suppressed by water stress.

Sub-Cellular Localisation of AtPK220 Proteins in Arabidopsis

Expression of a full length wild type AtPK220-GFP fusion protein intransgenic Arabidopsis was used to locate the sub-cellular localizationof the native protein. The primer pair SEQ ID NO:109 and SEQ ID NO:110produced a fragment that was digested with SmaI and BamHI to yield afragment comprising the full length open reading frame of AtPK220 and isdisclosed as SEQ ID NO:10 and cloned downstream, in frame with the greenfluorescence protein (GFP) in a pEGAD plasmid at the SmaI and BamHIsites. Additionally, the AtPK220 coding sequence was amplified usingprimer pair SEQ ID NO:198 and SEQ ID NO:199 and inserted upstream and inframe with GFP by AgeI digestion of pEGAD plasmid and the amplifiedAtPK220 fragment.

The 35S-GFP-AtPK220 and 35S-AtPK220-GFP constructs were transformed intoArabidopsis plants and homozygous transgenic plants (root tissues) wereused for visual screening of GFP signal under confocal microscope. Greenfluorescence was detected along plasma membrane, suggesting that AtPK220protein was associated with plasma membrane in roots and that AtPK220possibly functions as receptor kinase to sense or transduceenvironmental signals.

Isolation of BnPK220 from Brassica napus by 5′ and 3′ RACE

To isolate the homologous gene of AtPK220 from canola, a blast search(BLASTn) of NCBI Nucleotide Collection (nr/nt, est) and TIGR (DFCI)Brassica napus EST Database was done using AtPK220 sequence. Based onthe sequences with highest similarity, a pair of primers, SEQ ID NO:122and SEQ ID NO:123 were designed and used to PCR amplify a partialfragment of BnPK220. Both mRNA and genomic DNA isolated from Brassicaleaves were used as template for these amplifications. A DNA fragment ofabout 500 bp was obtained by PCR from canola genomic DNA template.Sequence analysis of this PCR product showed that it shares a highidentity with AtPK220 in nucleotide sequence as well in the intronorganisation.

Based on the partial sequence of BnPK220, 5′ and 3′ RACE was performedto isolate the full length BnPK220 cDNA. For 3′ RACE a forward primer,SEQ ID NO:124 and a nested primer, SEQ ID NO:125, were used. For 5′ RACEa reverse primer, SEQ ID NO:126, and its nest primer, SEQ ID NO:127,were designed. RACE-ready cDNA for either 5′ RACE or 3′ RACE was madefrom RNA isolated from young Brassica leaves.

The 5′ RACE yielded an amplified DNA of about 650 bp in length; and 3′RACE yielded a DNA of about 1 kb in size. Sequencing of these two RACEfragments showed high sequence similarity with AtPK220. A full-lengthmRNA of BnPK220 sequence was assembled by combining 5′RACE, partialBnPK220 fragment and 3′ RACE results.

A full length BnPK220 cDNA was amplified by RT-PCR using the PCR primersSEQ ID NO:128 and SEQ ID NO:129. This cDNA comprises an ORF of 1302nucleotides (SEQ ID NO:25) and encodes a protein of 433 amino acids (SEQID NO:26). Another full length BnPK220 cDNA was also amplified by theRT-PCR using cDNA made from B. napus. This cDNA (SEQ ID NO: 193) is98.6% identical to SEQ ID NO:25, and encodes a protein (SEQID NO:194) of99.3% identical to SEQ ID NO:26.

Isolation of Full-Length GmPK220 from Soybean by 5′ RACE

A Blastn search of NCBI EST database, a homolog of AtPK220 was found asa soybean (Glycine max) EST, CX709060.1. From this homolog, a unigenecluster of 13 ESTs was retrieved from a soybean EST database. A contigwas then assembled from these 13 ESTs, which covers a majority of thegene sequence.

The full-length sequence of GmPK220 (SEQ ID NO:41) was determined bycombining the assembled contig, 5′ RACE and 3′ RACE results. The 5′ RACEwas performed using the primers of SEQ ID NO:130 for primary RACE PCRand SEQ ID NO:131 for nested RACE PCR. The 3′ RACE was performed usingthe primers of SEQ ID NO:137 for primary RACE PCR and SEQ ID NO:138 fornested RACE PCR. GmPK220 encodes a protein as shown in SEQ ID NO:42.

Isolation of OsPK220 (Rice) Sequence by Database Mining

The rice genome (Oryza sativa, japonica cultivar) has been completelysequenced and is publically available. The homolog of AtPK220 in ricewas determined by BLAST search of a rice EST database and by BLASTPsearch of a genomic sequence database. The target having the highestscore was identified as Accession number Os05g0319700.

Os05g0319700 is abbreviated as OsPK220, and disclosed as SEQ ID NO:59,which encodes a protein disclosed as SEQ ID NO:60.

Isolation of ZmPK220 (Corn) Sequence

Two candidate homologs were found by BLAST search of the TIGR ESTdatabase, one a unigene Accession number TC333547 and the secondAccession number CO0439063.

Accession number TC333547 is 2125 nucleotides in length and contains anopen reading frame of 1377 nucleotides (SEQ ID NO:77) encoding a proteinof 458 amino acids (SEQ ID NO:78). This translated protein isfull-length and is larger than AtPK220 protein. The C-terminal kinasedomain is highly conserved between the Arabidopsis and corn proteinsequence, however, the N-terminal sequence is more variable.

CO439063 is a short EST sequence and is missing 5′ terminal sequence.The missing sequence was obtained by RACE methods. Two 5′ RACE primerswere designed based on the alignment between AtPK220 and CO439063. Theprimary 5′ RACE primer is SEQ ID NO:132 and the nested 5′ RACE primer isSEQ ID NO:133. The 3′ RACE was also performed using the primers of SEQID NO:139 for primary RACE PCR and SEQ ID NO:140 for nested RACE PCR.The ZmPK220 (SEQ ID NO:79) sequence was assembled based on 5′ RACE, 3′RACE results and CO439063 EST sequences. The corresponding proteinsequence was listed as SEQ ID NO:80.

Sequence analysis shows that CO439063 has higher sequence similaritywith rice OsPK220 than TC333547.

Isolation of BdPK220 Sequence from Brachipodium Distachyon (Bd)

Brachipodium is one of the model monocot plants for functional genomicresearch. A contig was assembled from public ESTs or GSSs, and it coversa 3′portion of BdPK220 according to homologue alignment. RACE usingBd81RAR1 primer (SEQ ID NO: 195) and Bd81RAR2 primer (SEQ ID NO: 196)designed from the contig and using Brachipodium leaf cDNA produced aunique fragment of about 650 bp. The assembling of the RACE sequence andthe contig gave the full length BdPK220 sequence (SEQ ID NO:24), whichencodes a protein of 461 amino acids (SEQ ID NO: 197).

Determination of GsPK220 (Cotton) Sequence by Database Mining

A BLAST search of a cotton (Gossypium) TIGR-EST database identified asequence cluster identified as Accession number TC79117, that has highsimilarity with AtPK220. This cluster has two overlapping ESTs, TC79117which is referred herein as GsPK220) and consists of an open readingframe of 1086 nucleotides (SEQ ID NO:81). The largest open reading frameencodes a protein of 361 amino acids (SEQ ID NO:82).

Drought Tolerant Phenotype of Hwell6 Mutant Found Under Water LimitedConditions and High Water Use Efficiency Under Both Drought and OptimalConditions

Two groups of plants were grown (5 plants per 3″ pot filled with thesame amount of soil-less mix) under optimal conditions in a growthchamber (22 C, 18 hr light, 150 uE, 70% relative humidity) until firstday of flower (n=6 per entry per treatment). At first flower all plantswere supplied with the same amount of water (optimal levels) but onegroup of plants was used for the optimal treatment and the other fordrought treatments. In the optimal treatment the pots were weighed dailyto determine daily water loss and then watered back up to optimallevels. In the drought treatment, pots were weighed daily to determinewater loss and allowed to dry out. Plants were harvested on days 0, 2and 4 of drought and optimal treatments for shoot biomassdeterminations. Lower water loss relative to shoot dry weight (DW) ascompared to control, under drought conditions indicates a droughttolerant phenotype. The ratio of shoot dry weight accumulated to waterlost during the treatment period provides a measure of water useefficiency (WUE). The hwe116 plants were delayed in flowering by 1 to 2days. Water loss relative to shoot biomass was significantly lower (by22%) in hwe116 than parent control under drought conditions. This resultindicates that the mutant is drought tolerant. It has also been foundthat under optimal conditions the water loss relative to shoot DW wasalso significantly lower in the mutant (by 41%) as compared to theparent control. This result is consistent with higher water useefficiency phenotype. Calculations of water use efficiency showed thatunder both drought (Table 1) and optimal (Table 2) conditions hwe116mutant uses water more efficiently because it accumulated more shootbiomass with less water (drought) or the same amount of biomass withless water (optimal).

TABLE 1 Water Use Efficiency (WUE) under drought conditions shoot DW WUEaccumulated - water lost - (g shootDW acc/kg Entry day 0 to 4 (g) day 0to 4 (g) water lost) hwe116 0.146 56.5 2.58 (+13%) Parent 0.134 58.62.28

TABLE 2 Water Use Efficiency (WUE) under optimal conditions shoot DWwater lost - WUE accumulated - day 0 (g shootDW acc/kg entry day 0 to 4(g) to 4 (g) water lost) hwe116 0.276 92.3 2.99 (+22%) Parent 0.271110.6 2.45

The final result of enhanced water use efficiency in the mutant isgreater shoot DW biomass as shown in Table 3 (harvested on day 4 from1^(st) flower).

TABLE 3 Final shoot DW biomass Drought - Optimal - shoot DW (g) shoot DW(g) entry Mean S.E. Mean S.E. hwe116 0.354 0.014 0.449 0.017 parent0.300 0.011 0.414 0.011 hwe116 as % of parent 118% 108%

The Hwe116 Mutant Maintains Higher Soil Water Content During DroughtTreatment, Reaches Water-Stress Conditions Later and Shows YieldProtection Following Drought Stress During Flowering Relative to ControlPlants.

An experiment was set up with 5 plants per 4″ pot filled with the sameamount of soilless mix. Two groups of plants (optimal and drought) weregrown under optimal conditions in a growth chamber (22 C, 18 hr light,150 uE, 70% relative humidity) until first day of flower (n=9 per entryand per group). At first flower all plants were supplied with the sameamount of water and further water was withdrawn for the drought treatedgroup of plants. The optimal group was watered daily as before. Pots inthe drought treated group were weighed daily for 6 days of treatment todetermine soil water content. After 6 days of drought treatment plantswere re-watered and allowed to complete their lifecycle as the optimalgroup under optimal conditions. At maturity the seeds were harvestedfrom each pot and the seed yield was determined for both optimal anddrought treated plants. The results of changes in soil water contentduring the drought treatments were determined. Soil water content wasmeasured as percentage of initial amount of water in the pot. Theresults indicate that the mutant was able to retain water in pots longerand therefore it reached the stress level (around 25% soil watercontent) 1 day later and wilted 1 day later than control. This treatmentcaused a yield reduction of 17% from optimal levels in the mutant,whereas in control the yield reduction was 41%. Therefore the mutantdemonstrated a yield protection of 24% relative to control, following adrought treatment.

The Hwe116 Mutant Seedlings Showed Less Sensitivity to Cold Stress.

Two groups of plants with 8 replicates per entry were grown with 3plants per 3″ pot under optimal conditions of 22° C. and short days toprolong vegetative growth and delay flowering (10 hr light 150 uE, and14 hr dark), 70% relative humidity in a growth chamber. At 10 days ofage (3 days post-transplanting of seedlings into soil from agar plates)the cold treatment group was placed in a chamber at 8° C. for 11 moredays of growth while the optimal group was maintained at 22° C. Plantswere harvested for shoot dry weight (DW) determinations at 21 days ofage. The results are shown in Table 4. The hwe116 mutant had smallerseedlings under optimal conditions than those of controls but after coldexposure the shoot DW was equivalent to that of the parent and aspercentage of the optimal DW it was higher than that of both controls by9 and 15% indicating that the growth of the mutant was not as inhibitedby cold as that of controls.

TABLE 4 shoot dry weight under optimal and cold conditions. optimal (22°C.) Cold (8° C.) shoot DW shoot DW (mg) (mg) shoot DW Entry Mean S.E.Mean S.E. % of optimal hwe116 6.65 0.30 2.85 0.13 43% parent 9.16 0.212.58 0.11 28% WT 9.30 0.20 3.18 0.21 34%The hwell6 Mutant has Thicker Leaves and Higher Chlorophyll Content PerLeaf Area. The Mutant Showed Delayed Leaf Senescence and Resistance toOxidative Stress.Plants were grown 1 per 3″ pot under optimal growth conditions in agrowth chamber (16 hr light, 300 uE, 22° C., 70% relative humidity).Early into flowering three leaf disks (86.6 um2 each) were taken fromthree youngest fully developed leaves and placed in petri dishescontaining filter paper with 5 uM N,N′-Dimethyl-4,4′-bipyridiniumdichloride (paraquat) solution as an oxidizing agent. Plates with leafdisks were placed under continuous light of 150 uE for 25 hours. Thisresulted in chlorophyll bleaching. The differences between the mutantand controls in the extent of bleaching were quantified by measuringchlorophyll content of the leaf disks. A leaf disk was also removed fromleaves that have not been exposed to paraquat treatment and optimalchlorophyll content was determined. These disks were also weighed. Theresults showed that the mutant had higher total chlorophyll content perleaf surface area (Table 5), however the leaves of this mutant arethicker (leaf disks were 15 to 24% heavier in the mutant compared tothose of controls). Chlorophyll content per gram of fresh leaf tissuewas, therefore, not different. There were no differences betweenchlorophyll a to b ratios between the mutant and controls. The hwe116mutant showed resistance to the oxidative stress as indicated by 5 to 7%higher chlorophyll content following paraquat treatment (Table 5). Leafsenescence was also delayed in the hwe116 mutant (data not shown).

TABLE 5 Effect of oxidative stress on chlorophyll content of leaves. 5uM paraquat Optimal in 24 hr light Chl (a + b)- Chl (a + b)- (mg/m2)(mg/m2) Entry Mean Std Err Mean Std Err % of opt hwe116 303.7 6.7 61.94.4 20% Parent 259.6 4.3 39.5 5.9 15% WT 250.2 5.7 32.1 2.9 13%

The Growth of Mutant Hwell6 Seedlings Showed Less Inhibition on LowNitrogen Containing Media.

Twelve seedlings were grown on an agar plate (6 plates per entry)containing ½ MS growth media with optimal (20 mM) or low (0.3 mM)nitrogen content. Plates were placed in a growth room with an 18 hrlight period (100 uE) for 6 days in a vertical position, then plateswere placed horizontally and seedlings were grown for another 4 daysbefore the shoots were harvested. The average seedling shoot DW after 10days of growth was calculated per plate. The results are shown in Table6. The shoot DW of hwe116 mutant grown under optimal conditions wassignificantly reduced but when grown on low nitrogen there were nodifferences. The shoot DW on low nitrogen in the mutant was 3 to 7%greater than in controls when compared to the optimal nitrogen levels.This indicates that the mutant may have better nitrogen use efficiency.

TABLE 6 Effect of nitrogen on seedling shoot DW Average seedling shootDW (mg) Optimal nitrogen Low nitrogen Entry Mean S.E. Mean S.E. % Opthwe116 1.03 0.03 0.23 0.01 22 Parent 1.34 0.04 0.20 0.01 15 WT 1.22 0.030.23 0.02 19

Knockout Mutant of PK220 Showed Drought Tolerant Trends and Higher WaterUse Efficiency Under Drought Treatment.

Plant lines obtained from the SALK institute that were T-DNA knockoutsin the AtPK220 gene (SALK_147838) were grown (5 per 3″ pot) underoptimal conditions in a growth chamber (18 hr light, 150 uE, 22° C., 60%relative humidity) until first open flower (n=8 per entry and perharvest). The drought treatment was started by watering all plants withthe same amount of water and cessation of further watering. Pots wereweighed daily and plants were harvested for shoot DW determinations ondays 0, 2 and 4 of the drought treatment. The result showed that waterlost from pots in 2 days relative to shoot DW on day 2 was significantlylower (by 13%) for the knockout mutant and its shoot DW was alsosignificantly greater (by 24%) on day 2 as compared to controlwild-type. This result is consistent with drought tolerant phenotype.

The results showed that the water use efficiency of the knockout mutantwas greater than that of the control-WT as the knockout mutant was ableto accumulate more shoot biomass in the 2 days of treatment while usingthe same amount of water as control (Table 7).

TABLE 7 Water use efficiency under drought treatment WUE entry g waterlost g shoot DW gain (g shoot/kg water) PK220- 43.1 0.059 1.37 knockoutWT 42.9 0.035 0.82

Transgenic Lines of 35S-HP-At(270)PK220 Construct in Arabidopsis ShowedDrought Tolerance.

Plants were grown (5 per 3″ pot and 8 pots per entry per harvest) underoptimal conditions in a growth chamber (18 hr light, 150 uE, 22° C., 60%relative humidity) until first day of flower. The drought treatment wasstarted by watering all pots with the same amount of water and cessationof further watering. Pots were weighed daily for water lossdeterminations and plants were harvested for shoot biomass on day 4 ofdrought treatment. The results (Table 8) showed that 11 out of13transgenic lines demonstrated a drought tolerant phenotype (having alower water loss over 2 days relative to shoot biomass on day 4). Fourof the lines showed a slight delay in flowering (1 day), as did thehwe116 mutant. The final shoot biomass on day 4 was greater for most ofthe transgenic lines as compared to control WT. These results areindicative of a drought tolerant phenotype in the transgenic linesdown-regulated in PK220 expression. As examples, the reduction inexpression level of AtPK220 for the top 3 performing lines: 65-4, 38-5,and 59-3, are 75%, 47% and 58%.

TABLE 8 Drought tolerance and shoot DW (day 4) for 35S-HP-At(270)PK220transgenic lines relative to wild type (WT) and the hwe116 mutantrelative to parent control. drought tolerance shoot DW entry % ofcontrol % of control 65-4 119% 132% 38-5 116% 124% 59-3 112% 119% 33-7111% 114%  54-11 108% 115% 56-3 107% 115%  43-11 107% 113% 23-8 106%111% 12-2 106% 110% 63-4 104% 110% 32-1 104% 109% 30-3 101% 104% 74-2101% 107% WT 100% 100% hwe116 186% 106% parent 100% 100%Drought Tolerance of 35S-HP-At(270)PK220 Transgenic Lines in Arabidopsisand Enhanced Water Use Efficiency were Confirmed.

The transgenic lines of 35S-HP-At(270)PK220 were grown with 5 per 3″ potunder optimal conditions in a growth chamber (18 hr light, 150 uE, 22°C., 60% relative humidity) until first flower (n=8). Drought treatmentwas started at first flower by watering all the pots with the sameamount of water and cessation of further watering. The pots were weigheddaily for the 4 days of drought treatment and plants were harvested ondays 0, 2 and 4 of treatment. The results confirmed that water lost in 2days relative to shoot biomass on day 2 was lower in five transgeniclines relative to controls, confirming their drought tolerant phenotype(Table 9). The shoot DW on day 2 was greater in 5 of the transgeniclines.

TABLE 9 Drought tolerance and shoot DW for 35S-HP-At(270)PK220transgenic lines drought tolerance shoot DW entry % of WT % of WT 59-3110% 105% 65-4 110% 98% 38-5 107% 109% 33-7 103% 106% 56-3 102% 95% 54-11 101% 103% null (65-1) 99% 99% WT 100% 100%

The water use efficiency was greater than that of controls during the 4days of drought treatment for three transgenic lines and this enhancedwater use efficiency was due to greater shoot DW accumulation (Table10).

TABLE 10 Water use efficiency between day 0 and 4 of the droughttreatment in transgenic lines of 35S-HP-At(270)PK220. WUE shoot DW (gshoot/kg accumulated (g) water lost (g) water) entry d0-d4 d0 to d4 d0to d4 65-4 0.090 62.5 1.44 (+22 to 33%) 12-2 0.079 62.2 1.27 (+7 to 17%)56-3 0.079 62.7 1.25 (+6 to 16%) null (65-1) 0.068 62.7 1.08 WT 0.07361.9 1.18

Transgenic Lines of 35S-HP-At(270)PK220 in Arabidopsis had Lower WaterLoss Relative to Shoot Biomass and Enhanced WUE Under OptimalConditions.

Plants of 35S-HP-At(270)PK220 transgenic lines 65-7 and 59-5, WTColumbia, hwe116 mutant and its parent were grown (5 per 3″ pot) underoptimal conditions in a growth chamber (22° C., 18 hr light—200 uE, 60%relative humidity) until first flower (n=8 per entry, per harvest). Atfirst flower all pots in the water limited group were watered with thesame amount of water (to a pot weight of 120 g in first 4 days and to130 g for last 3 days (as plants grew larger they required more water).Pots were weighed daily to determine daily water loss and plants wereharvested on day 0 and day 7 of this treatment. Water use efficiency(WUE) was calculated from the ratio of shoot biomass accumulated towater lost. The results are shown in Table 11.

TABLE 11 Water Use Efficiency under optimal conditions WUE shoot DW (gshoot/kg accumulated (g) water lost (g) water) entry d0-d4 d0 to d4 d0to d4 59-5 0.514 223 3.31 (+4%) 65-4 0.671 276 2.43 (+9%) WT 0.517 2322.23 hwe116 0.420 191 2.19 (5%) parent 0.421 202 2.08The results show that under optimal water conditions the two transgeniclines and the mutant had enhanced water use efficiency.Growth Rates of the 35S-HP-At(270)PK220 Transgenic Arabidopsis wereGreater than Those of Controls During Both Optimal and Water LimitedConditions.

Plants of 35S-HP-At(270)PK220 transgenic line 65-4 and WT Columbia weregrown (5 per 3″ pot) under optimal conditions in a growth chamber (22°C., 18 hr light—150 uE, 60% relative humidity) until first flower (n=8per entry, per treatment and per harvest). At first flower all pots inthe water limited group were watered with the same amount of water (to apot weight of 95 g), and further watering was stopped for 2 days. Ittook 2 days for the water limited group of plants to reach about 30% ofinitial soil water content (about 55 g total pot weight), referred to aspre-treatment. At that time the water limited treatment was deemed tohave started (day 0 of treatment) and plants were watered daily up to atotal pot weight of 55 g for 3 days, and up to 65 g in the following 4days (until day 7 of treatment). The optimal group was maintained underoptimal conditions by watering the pots daily up to 100 g total potweight in the 2 pre-treatment days, the first 3 days of treatment andthen up to 130 g in the last 4 days of treatment (as plants grew largerthey required more water). The daily water loss from the pots wasmeasured for all the plants and plants in both groups were harvested ondays 0, 1, 2, 3, 5, and 7 of treatment for shoot dry weightdeterminations. The water loss relative to the shoot biomass (droughttolerant phenotype) was calculated over the initial two days before thestart of treatment, during the first 3 days of treatment and during thelast 4 days of treatment. The results under both optimal (Table 12) andwater limited (Table 13) conditions are shown. The transgenic line 65-4lost less water relative to shoot biomass than WT in both optimal andwater limited conditions. Under limited water conditions this isconsistent with enhanced drought tolerance phenotype.

TABLE 12 Water loss in g/shoot DW in g under optimal conditions. Entrypre-treatment d0-d3 d3-d7 65-4 231 ± 9 162 ± 3 237 ± 5 WT 275 ± 8 178 ±7 243 ± 6

TABLE 13 Water loss in g/shoot DW in g and Drought tolerance (aspercentage of WT) under water limited conditions. pre-treatment d0-d3d3-d7 (drought toler. in (drought toler. in (drought toler. in Entry %of WT) % of WT) % of WT) 65-4 174 ± 2 (108%) 83 ± 2 (115%) 153 ± 6(113%) WT 189 ± 4 (100%) 97 ± 4 (100%) 175 ± 4 (100%)

Growth rates of the plants were calculated over the seven days of bothtreatments. The results showed that transgenic line 65-4 had largerplants (up to 24%) than the wild type throughout the treatment underboth conditions. The growth rate (shoot dry weight accumulated per dayover the 7 days of treatment) was slightly greater for the transgenicline under both optimal and water limited conditions (63.3 and 21.3 mgshoot/day, respectively) than that of WT control (58.3 and 20.4 mgshoot/day, respectively).

The Transgenic Line of 35S-HP-At(270)PK220 Arabidopsis and the hwell6Mutant Grow Better Under Limited Nitrogen Conditions than Controls.

The 35S-HP-At(270)PK220 transgenic line 65-5, its segregated nullcontrol (null 65-1) and wild-type (WT) plus the hwe116 mutant and itsparent control were analyzed for growth characteristics of youngseedling under optimal and limited nitrogen conditions. Nitrogen contentrefers to the available nitrogen for plant growth, including nitrate andammonium sources. Seedlings were grown on agar plates (10 per plate and5 plates per entry and per treatment) containing either optimalnutrients (including 20 mM nitrogen) or low (limiting to growth)nitrogen (optimal all nutrients except for nitrogen being 0.5 mM).Plates were placed in a growth chamber at 18 hr lights of 200 uE and 22°C. Seedlings were grown for 14 days before being harvested for shootbiomass (8 seedlings) and chlorophyll determinations (2 seedlings). Onoptimal plates there were no differences in average seedling shootbiomass except for the hwe116 mutant, as shown before had slightlysmaller seedling shoot DW (not significant). On low nitrogen the hwe116mutant had significantly bigger seedling shoot DW and showed 30% lessinhibition in growth as compared to its parent. The transgenic line 65-5showed slightly greater shoot DW than controls and was 5% to 7% lessinhibited in growth than the controls (Table 14).

TABLE 14 Effect of nitrogen on seedling shoot DW Average seedling shootDW (mg) Optimal N (20 mM) Low N (0.5 mM) entry Mean Std Err Mean Std Err% of opt 65-5 5.3 0.1 2.9 0.1 56% WT 5.5 0.3 2.8 0.1 51% hwe116 4.8 0.23.8 0.3 80% parent 5.1 0.2 2.6 0.1 50%The total chlorophyll content of seedling shoots grown under low Nlevels reflected the shoot DW results. Chlorophyll content is veryclosely linked to available N and one of the major symptoms ofN-deficiency in plants is leaf chlorosis or bleaching. Table 15 showsthat chlorophyll content of the transgenic line 65-5 and the mutanthwe116 was reduced less than that of the controls.

TABLE 15 Effects of nitrogen on seedling shoot total chlorophyll contentseedling shoot chlorophyll content (ug/g) Optimal N (20 mM) Low N (0.5mM) entry Mean Std Err Mean Std Err % of opt 65-5 902 35 244 22 27% WT854 102 156 17 18% hwe116 1006 51 376 37 37% parent 836 59 208 47 25%

These results confirmed that the hwe116 mutant grew better on limitednitrogen and the transgenic line showed the same trends. Therefore,down-regulation of the PK220 gene in plants appears to result inincreased nitrogen use efficiency (accumulation of more biomass per unitof available nitrogen).

The Transgenic Line of 35S-HP-At(270)PK220 Arabidopsis and the hwell6Mutant Germinate Faster and have Higher Rates of Germination in theCold.

Germination under cold (10° C.) conditions was assessed in thetransgenic line 65-5 carrying the 35S-HP-At(270)PK220 construct relativeto WT-control and that of the hwe116 mutant relative to its parentalcontrol on agar plates containing optimal growth media. Four plates perentry with 30 seeds each were prepared and placed in the chamber at 10°C., 18 hr light (200 uE). Germination (emergence of the radicle) scoredas a percentage of viable seeds, was noted twice daily for 5 daysstarting with day 5 from placing of seeds on plates (no germinationbefore day 5). Once no further changes were observed in germination allplates were placed in a chamber at 22° C. to check for viability of theseeds that had not germinated. All entries showed 98 to 100% seedviability, the hwe116 mutant had 94%. viabilty. The results of thegermination assessment at 10° C. (Table 16) indicate that the transgenicline 65-5 germinated sooner than it's WT-control. The hwe116 mutant hadhigher rates of germination in the cold than its parent control. Thesedata, together with the evidence that the mutant grows better under coldconditions are indicative of a greater seed and seedling vigor undercold stress

TABLE 16 percentage germination of viable seeds at 10° C. Hours @ 10° C.% Viable entry #reps 114.5 121 139 145 163 169 188.5 212.5 235 241 Seed65-5 4 15.1 32.8 75.7 80.7 90.0 90.8 90.8 92.5 93.3 94.2 99.2 WT 4 5.916.0 55.4 62.9 78.1 79.0 80.7 80.7 81.5 81.5 98.4 hwe116 4 15.9 28.467.5 81.4 94.2 98.0 99.0 99.0 100.0 100.0 94.0 Parent 4 6.7 26.7 72.577.5 85.0 85.0 85.9 85.9 85.9 85.9 100.0

Gas Exchange Measurements Support Higher WUE in Transgenic35S-HP-At(270)PK220 Arabidopsis Under Optimal Conditions

Plants of two transgenic lines and WT were grown in four inch diameterpots (one per pot) under optimal conditions in a growth chamber at 18 hrlight (200 uE), 22° C., 60% RH. Eight days from first open flower gasexchange measurements were made on the youngest, fully developed leaf of10 to 11 replicates per entry. Photosynthesis and transpiration rateswere measured inside the growth chamber at the ambient growth light andtemperature conditions and 400 ppm carbon dioxide using Li-6400 andArabidopsis leaf cuvette. From the ratio of photosynthesis totranspiration instantaneous water use efficiency (WUE) was calculated.The results are shown in Table 17. The WUE in the transgenic lines was11 and 18% greater than that of the WT. This data is consistent with theWUE measurements over a period of few days using the ratio of biomassaccumulated to water lost in transpiration.

TABLE 17 Photosynthesis (umol carbon dioxide/m2/s), transpiration (mmolH2O/m2/s) and WUE measured under optimal growth conditions. Phots.Trans. WUE (umol/ Photos. (mmol/ Trans. (Photos/ WUE entry m2/s) (% WT)m2/s) (% WT) Trans) (% WT) 59-6 3.9 ± 0.2 105% 4.2 ± 0.5 95% 1.03 ± 0.11118% 65-5 3.6 ± 0.2 97% 3.8 ± 0.4 86% 0.97 ± 0.13 111% WT 3.7 ± 0.2 4.4± 0.2 0.87 ± 0.05

Drought Tolerance of 35S-HP-At(270)PK220 Transgenic Arabidopsis Resultsin Seed Yield and Biomass Protection Following Drought Stress.

Plants of two transgenic lines and the WT were grown (5 per 3 inch potcontaining equal amount of soil) under optimal conditions in a growthchamber (22 C, 18 hr light of 200 uE, 60% RH) until first open flower.At first flower the drought treatment was applied to half of the plantswhile the other half was maintained under optimal conditions untilmaturity. The drought treatment consisted of watering all the plants tothe same saturated water level. Plants were then weighed daily tomonitor water loss from the pots and their water content was equalizeddaily by watering all pots to the level of the heaviest pot. As a resultthe soil water content was declining and reached stress levels withplants wilting on day 4. Plants were maintained at that stress level foranother 2 days and on day 6 all plants were re-watered and maintainedunder optimal conditions for the rest of their life cycle. At maturityboth optimal and drought plants were harvested for seed and shootbiomass. The impact of drought stress on both seed yield and shootbiomass was determined by comparing the optimal and drought treatedplants. The results are shown in Table 18. Under optimal conditions theseed yield and the final shoot biomass of the transgenic lines was 7 to10% higher than that of the WT. Following the drought stress duringflowering the reduction in seed yield and the shoot biomass were not asgreat in transgenic plants as in the WT, resulting in seed yieldprotection of 5-7% and shoot biomass protection of 4%. The protectionwas calculated as the difference between the transgenics and WT in seedyield or shoot biomass a percentage of optimal.

TABLE 18 Seed yield and final shoot biomass from optimal and droughtstressed plants, n = 10 Seed yield - Shoot DW - drought % of Shoot DW -% of entry Seed yield - opt (g) opt (g) (g) opt drought (g) opt 59-61.29 ± 0.05 2.96 ± 0.13 1.06 ± 0.03 82% 2.37 ± 0.07 80% 65-5 1.27 ± 0.032.89 ± 0.08 1.01 ± 0.02 80% 2.32 ± 0.06 80% WT 1.18 ± 0.04 2.69 ± 0.100.89 ± 0.02 75% 2.04 ± 0.05 76%

Over-Expression of Wild Type AtPK220 in Hwe116.2 Background can Restorethe WT Phenotype

Transgenic plants of 35S-AtPK220 (in hwe116.2) were grown (5 per 3 inchpot) under optimal conditions in a growth chamber as described aboveuntil the first open flower. Drought treatment was applied by wateringall plants to the same saturated level. Further watering was withheld.Plants were weighed daily to determine the daily water loss and allplants were harvested on day 4 of treatment by which time all plantsshowed wilting. The water loss relative to final shoot biomass was usedto calculate drought tolerance where that of WT was assumed at 100%. Thedata are shown in Table 19. Three transgenic lines showed a reduction indrought tolerance from the mutant levels as indicated by increased waterloss relative to shoot biomass. The three transgenic lines also floweredearlier than the mutant line and similar to the time that the WT linesflowered. These results support the conclusion that the AtPK220 genemutation in hwe116.2 is responsible for the altered phenotypes observedand expression of a WT gene restore the WT characteristics of a mutantplant.

TABLE 19 Water loss relative to shoot biomass and drought tolerance, n =8 Days to Water lost in 3 d/shoot DW Drought tolerance entry flower d4(% of WT) 28-4  20.9 ± 0.1 155.1 ± 3.1 111% 2-4 21.8 ± 0.1 164.7 ± 2.4105%  7-11 21.6 ± 0.1 177.9 ± 4.4 97% hwe116.2 23.1 ± 0.2 134.9 ± 3.6117% WT 20.8 ± 0.2 173.4 ± 5.1 100%Down Regulation of AtPK220 with the AtPK220-Promoter (P_(PK)) inArabidopsis Results in Enhanced Drought Tolerance of Plants

Arabidopsis plants of P_(PK)-HP-At(270)PK220 were grown (5 per 3 inchpot) under optimal conditions in a growth chamber as mentioned aboveuntil the first open flower. Drought treatment was applied then bywatering all plants to the same saturated level. Further water waswithheld. Plants were weighed daily to determine the daily water lossand all plants were harvested on day 4 of treatment (all plants werewilted). The water loss relative to final shoot biomass was used tocalculate drought tolerance where that of WT was assumed at 100%. Theresults of this study are shown in Table 20.

TABLE 20 Water loss relative to shoot biomass and drought tolerance, n =8 Drought Water lost in tolerance entry Days to flower 3 d/shootDW d4 (%WT) 14-04 22 158 ± 5 116% 15-06 20 183 ± 8 104% 45-3  20 185 ± 9 103% WT20 190 ± 9 100%One of the transgenic lines, 14-04, showed significantly greater droughttolerance than the wild type control as indicated by lower water lossrelative to shoot biomass. This result is supported by data from line14-04 that showed nearly complete inhibition of PK220 gene expression.The expression of AtPK220 was reduced by nearly 96% in the rootscompared to WT. These results indicate that down regulation of PK220 inthe roots is sufficient to achieve significant drought tolerancephenotype and presumably enhanced water use efficiency.Overexpression of Brassica napus PK220 in the Arabidopsis hwe116 Mutantcan Restore the WT PhenotypeTransgenic plants of 35S-BnPK220 (in hwe116) plus two null controls(segregated siblings of the transgenic lines without the transgene,therefore hwell6 mutant) were grown (5 per 3 inch pot) under optimalconditions in a growth chamber as mentioned above until the first openflower. Drought treatment was applied then by watering all plants to thesame saturated level. Further water was withheld. Plants were weigheddaily to determine the daily water loss and all plants were harvested onday 4 of treatment (all plants were wilted). The water loss relative tofinal shoot biomass was used to calculate drought tolerance where thatof WT was assumed at 100%. The results of this study are shown in Table21. The results indicate that 6 lines had a reduction of 8% or more indrought tolerance as compared to the nulls (the hwe116 mutantbackground) and therefore restoration towards the WT phenotype. Thisindicates that BnPK220 is functional and can work in the Arabidopsis.

TABLE 21 Water loss relative to shoot DW and drought tolerance, n = 8Water lost in Drought tolerance entry 3 d/shoot DW d4 (% of null) 106-11148 ± 6 98% 67-6 150 ± 4 97% 51-6 152 ± 4 96%  5-1 152 ± 2 95%  74-12157 ± 5 92% 38-7 160 ± 5 90% 70-2 161 ± 2 89% 97-3 164 ± 5 87% 31-6 165± 4 87% 93-8 172 ± 4 82% Null 38-10 146 ± 3 100% Null 90-7 135 ± 5 107%

Transgenic Brassica Lines Having a 35S-AtPK220L292F Construct ShowedDrought Tolerance and Higher Water Use Efficiency

Down regulation of endogenous PK220 activity was demonstrated using adominant negative strategy by expression of the mutant allele of theAtPK220 gene in Brassica napus. Three Brassica napus transgenic lineshaving the Arabidopsis mutant AtPK220L292F gene and one null controlline (a segregated sibling of the transgenic line lacking the transgene)per line were grown in 4.5 inch diameter pots containing equal amountsof soilless mix (Sunshine Professional Organic Mix #7) under optimalconditions of 16 hr light (400 uE) and 22 C day/18 C night temperature.At the four leaf stage, two treatments were applied. In the optimaltreatment plants were watered to saturation and pots were covered withplastic bags to prevent any water loss from the pots due to evaporation.These plants were weighed daily for 7 days to determine the water lossfrom the pots due to transpiration and the same amount of water wasadded back daily to each pot to maintain the plants under optimal waterconditions. In the drought treatment all plants were watered tosaturation levels. Pots were covered with plastic and were weigheddaily. However, these pots were watered daily to the level of theheaviest pots. This treatment went for 7 days with the soil watercontent gradually reaching stress levels. Plants started to wilt by day5. At the end of the 7 days both groups of plants were harvested forshoot biomass determinations.

Gas exchange measurements were done on drought treated plants of twotransgenic lines plus their nulls on days 3 and 4 of the treatment.Photosynthesis and transpiration were measured on leaf 3 under steadystate growth conditions of 400 uE light, 400 ppm carbon dioxide and 22 Cusing Li-6400. From the ratio of photosynthesis to transpiration, wateruse efficiency (WUE) was calculated. The drought treated plants wereused to calculate the drought tolerance (as percentage of their nulls).This was done using the ratio of cumulative daily transpirational waterloss between days 3 and 7, relative to the final shoot dry weight andnormalizing it to the nulls (set at 100%).

The results in Table 22 indicate that transgenic lines had strong trendstoward greater drought tolerance. This was a result of lower water lossrelative to shoot dry weight, a phenotype present also under optimalconditions.

The gas exchange data (Table 23) showed that on both days 3 and 4 of thedrought treatment the transgenic plants had slightly higher WUE thancontrols (4 to 16%).

Water use efficiency calculated from the ratio of photosynthesis totranspiration provides only a single point, instantaneous measurementrather than cumulative measurement over the period of treatment and as aresult may be of lesser magnitude.

In conclusion, the data with transgenic 35S-AtPK220L292F Brassica plantsindicate that water use efficiency technology is transferable toBrassica when using a AtPK220L292F gene from a heterologous species.

TABLE 22 Water loss between days 3 and 7 relative to final shoot dryweight under optimal and drought treatment. Drought tolerance (% of theappropriate null). n = 8 optimal - g water lost drought - g water lostDrought tolerance entry d3-7/g shootDW d7 d3-7/g shootDW d7 (% of null)Tr-05 172 ± 6 121 ± 5 109% Null-05 190 ± 4 133 ± 7 100% Tr-27 194 ± 6134 ± 7 113% Null-27 205 ± 8  155 ± 11 100% Tr-09  171 ± 10 129 ± 3 113%Null-09  178 ± 17  149 ± 13 100%

TABLE 23 Photosynthesis (umol carbon dioxide/m2/s), Transpiration (mmolH2O/m2/s) and WUE (Photos/Trans) on days 3 and 4 of drought treatment. n= 8 Photos Trans. WUE Photos. Trans. WUE entry D3 D3 D3 D4 D4 D4 Tr-0513.4 ± 1.2 2.1 ± 0.2 6.6 ± 0.2 11.6 ± 1.3 1.9 ± 0.2 6.1 ± 0.4 (116% ofnull) (104% of null) Null-05 14.4 ± 1.1 2.5 ± 0.2 5.7 ± 0.2 12.6 ± 1.22.2 ± 0.2 5.9 ± 0.3 Tr-27 14.1 ± 0.7 2.4 ± 0.2 5.9 ± 0.3 11.4 ± 1.6 1.9± 0.3 6.2 ± 0.5 (105% of null) (108% of null) Null-27 14.1 ± 1.3 2.5 ±0.1 5.6 ± 0.5 13.7 ± 1.0 2.4 ± 0.1 5.7 ± 0.4

Sequence ID Reference Chart

SPECIES SEQ ID NO: REFERENCE ARABIDOPSIS THALIANA SEQIDNO: 1 AtPK220 NT1299 ARABIDOPSIS THALIANA SEQIDNO: 2 AtPK220 AA 432 ARABIDOPSIS THALIANASEQIDNO: 3 AtPK220L292F NT 1299 ARABIDOPSIS THALIANA SEQIDNO: 4AtPK220L292F AA 432 ARABIDOPSIS THALIANA SEQIDNO: 5 AtPK220L292F_partialNT 1160 ARABIDOPSIS THALIANA SEQIDNO: 6 AtPK220L292F_partial_orf AA 383ARABIDOPSIS THALIANA SEQIDNO: 7 AtPK220_partial NT 1160 ARABIDOPSISTHALIANA SEQIDNO: 8 AtPK220_partial_orf AA 383 ARABIDOPSIS THALIANASEQIDNO: 9 AtPK220_with_UTR NT 1542 ARABIDOPSIS THALIANA SEQIDNO: 10AtPK220_for_35s-AtPK220 NT 1309 ARABIDOPSIS THALIANA SEQIDNO: 11AtPK220_partial NT 1177 ARABIDOPSIS THALIANA SEQIDNO: 12 At(150)PK NT154 ARABIDOPSIS THALIANA SEQIDNO: 13 At(270)PK NT 288 ARABIDOPSISTHALIANA SEQIDNO: 14 AtPK220_promoter NT 1510 ARABIDOPSIS THALIANASEQIDNO: 15 At4g32000_UTR NT 157 ARABIDOPSIS THALIANA SEQIDNO: 16At4g32000 NT 1257 ARABIDOPSIS THALIANA SEQIDNO: 17 At4g32000 AA 418ARABIDOPSIS THALIANA SEQIDNO: 18 At5g11020 NT 1302 ARABIDOPSIS THALIANASEQIDNO: 19 At5g11020 AA 433 ARABIDOPSIS THALIANA SEQIDNO: 20 At2g25440NT 2016 ARABIDOPSIS THALIANA SEQIDNO: 21 At2g25440 AA 671 ARABIDOPSISTHALIANA SEQIDNO: 22 At2g23890 NT 1662 ARABIDOPSIS THALIANA SEQIDNO: 23At2g23890 AA 553 BRACHYPODIUM SEQIDNO: 24 BdPK220 NT 1386 DISTACHYONBRASSICA NAPUS SEQIDNO: 25 BnPK220 NT 1302 BRASSICA NAPUS SEQIDNO: 26BnPK220 AA 433 CICHORIUM ENDIVIA SEQIDNO: 27 EL362007.1 NT 657 CICHORIUMENDIVIA SEQIDNO: 28 EL362007.1_ORF AA 218 CITRUS CLEMENTINA SEQIDNO: 29CX290402.1 NT 474 CITRUS CLEMENTINA SEQIDNO: 30 CX290402.1_ORF AA 157CITRUS SINENSIS SEQIDNO: 31 CK934154.1 NT 770 CITRUS SINENSIS SEQIDNO:32 CK934154.1_ORF AA 257 COFFEA CANEPHORA SEQIDNO: 33 DV708241.1 NT 621COFFEA CANEPHORA SEQIDNO: 34 DV708241.1_ORF AA 206 EUCALYPTUS GUNNIISEQIDNO: 35 CT986101.1 NT 411 EUCALYPTUS GUNNII SEQIDNO: 36CT986101.1_ORF AA 136 FESTUCA ARUNDINACEA SEQIDNO: 37 DT714073 NT 522FESTUCA ARUNDINACEA SEQIDNO: 38 DT714073_ORF AA 173 GINKGO BILOBASEQIDNO: 39 EX942240.1 NT 740 GINKGO BILOBA SEQIDNO: 40 EX942240.1_ORFAA 247 GLYCINE MAX SEQIDNO: 41 GmPK220 NT 1254 GLYCINE MAX SEQIDNO: 42GmPK220 AA 418 HELIANTHUS SEQIDNO: 43 EE622910.1 NT 702 ARGOPHYLLUSHELIANTHUS SEQIDNO: 44 EE622910.1_ORF AA 233 ARGOPHYLLUS HELIANTHUSCILIARIS SEQIDNO: 45 EL429543.1 NT 752 HELIANTHUS CILIARIS SEQIDNO: 46EL429543.1_ORF AA 251 HELIANTHUS EXILIS SEQIDNO: 47 EE654885.1 NT 630HELIANTHUS EXILIS SEQIDNO: 48 EE654885.1_ORF AA 209 HORDEUM VULGARESEQIDNO: 49 TC151622 NT 780 HORDEUM VULGARE SEQIDNO: 50 TC151622_ORF AA259 IPOMOEA BATATAS SEQIDNO: 51 EE883089.1 NT 816 IPOMOEA BATATASSEQIDNO: 52 EE883089.1_ORF AA 272 LACTUCA SATIVA SEQIDNO: 53 DW125133.1NT 867 LACTUCA SATIVA SEQIDNO: 54 DW125133.1_ORF AA 288 MEDICAGOTRUNCATULA SEQIDNO: 55 Contig NT 804 MEDICAGO TRUNCATULA SEQIDNO: 56Contig AA 267 NICOTIANA TABACUM SEQIDNO: 57 BP131484.1 NT 636 NICOTIANATABACUM SEQIDNO: 58 BP131484.1 AA 211 ORYZA SATIVA SEQIDNO: 59NM_001061720.1 NT 1437 ORYZA SATIVA SEQIDNO: 60 NP_001055185.1 AA 478PHYSCOMITRELLA SEQIDNO: 61 EDQ75046.1_cds NT 891 PHYSCOMITRELLA SEQIDNO:62 EDQ75046.1 AA 297 PICEA SEQIDNO: 63 TC12392 NT 1065 PICEA SEQIDNO: 64TC12392_orf AA 354 PINUS SEQIDNO: 65 CT578985.1 NT 596 PINUS SEQIDNO: 66CT578985.1_ORF AA 199 POPULUS SEQIDNO: 67 TC76879 NT 1377 POPULUSSEQIDNO: 68 TC76879_ORF AA 459 SACCHARUM SEQIDNO: 69 TC46535 NT 693OFFICINARUM SACCHARUM SEQIDNO: 70 TC46535_ORF AA 230 OFFICINARUMTRIPHYSARIA SEQIDNO: 71 DR169688.1 NT 414 VERSICOLOR TRIPHYSARIASEQIDNO: 72 DR169688.1_ORF AA 137 VERSICOLOR TRITICUM AESTIVUM SEQIDNO:73 TC254793 NT 1140 TRITICUM AESTIVUM SEQIDNO: 74 TC254793_ORF AA 380VITIS VINIFERA SEQIDNO: 75 CAO44295.1_cds NT 978 VITIS VINIFERA SEQIDNO:76 CAO44295.1 AA 325 ZEA MAYS SEQIDNO: 77 TC333547 NT 1377 ZEA MAYSSEQIDNO: 78 TC333547_ORF AA 458 ZEA MAYS SEQIDNO: 79 ZmPK220 NT 1188 ZEAMAYS SEQIDNO: 80 ZmPK220 AA 396 GOSSYPIUM SEQIDNO: 81 TC79117 NT 1086GOSSYPIUM SEQIDNO: 82 TC79117_ORF AA 361 SOLANUM SEQIDNO: 83 Contig3 NT1089 LYCOPERSICUM AQUILEGIA SEQIDNO: 84 DR918821 NT 875 AQUILEGIASEQIDNO: 85 DR918821_ORF AA 292 CENTAUREA MACULOSA SEQIDNO: 86EL933228.1 NT 696 CENTAUREA MACULOSA SEQIDNO: 87 EL933228.1_ORF AA 231CICHORIUM INTYBUS SEQIDNO: 88 EH693146.1 NT 842 CICHORIUM INTYBUSSEQIDNO: 89 EH693146.1_ORF AA 281 CUCUMIS MELO SEQIDNO: 90 AM742189.1 NT495 CUCUMIS MELO SEQIDNO: 91 AM742189.1_ORF AA 164 ERAGROSTIS CURVULASEQIDNO: 92 EH186232.1 NT 375 ERAGROSTIS CURVULA SEQIDNO: 93EH186232.1_ORF AA 124 GERBERA HYBRID SEQIDNO: 94 AJ753651.1 NT 414GERBERA HYBRID SEQIDNO: 95 AJ753651.1_ORF AA 137 HELIANTHUS PARADOXUSSEQIDNO: 96 EL488199.1 NT 498 HELIANTHUS PARADOXUS SEQIDNO: 97EL488199.1_ORF AA 165 IPOMOEA NIL SEQIDNO: 98 BJ566706.1 NT 612 IPOMOEANIL SEQIDNO: 99 BJ566706.1_ORF AA 203 NUPHAR ADVENA SEQIDNO: 100DT603238.1 NT 708 NUPHAR ADVENA SEQIDNO: 101 DT603238.1_ORF AA 235SYNTHETIC PRIMER SEQIDNO: 102 747F NT 30 SYNTHETIC PRIMER SEQIDNO: 103747R NT 34 SYNTHETIC PRIMER SEQIDNO: 104 C747F2 NT 32 SYNTHETIC PRIMERSEQIDNO: 105 C747R2 NT 31 SYNTHETIC PRIMER SEQIDNO: 106 A220BamF1 NT 42SYNTHETIC PRIMER SEQIDNO: 107 A220PstR NT 40 SYNTHETIC PRIMER SEQIDNO:108 K188R NT 30 SYNTHETIC PRIMER SEQIDNO: 109 A220A1SmaF2 NT 53SYNTHETIC PRIMER SEQIDNO: 110 A220BamR NT 38 SYNTHETIC PRIMER SEQIDNO:111 A220SmaF NT 41 SYNTHETIC PRIMER SEQIDNO: 112 A220BamF2 NT 41SYNTHETIC PRIMER SEQIDNO: 113 A220XbaR NT 39 SYNTHETIC PRIMER SEQIDNO:114 K116SacF NT 35 SYNTHETIC PRIMER SEQIDNO: 115 K270SacR NT 37SYNTHETIC PRIMER SEQIDNO: 116 K116BamF NT 37 SYNTHETIC PRIMER SEQIDNO:117 K270XbaR NT 40 SYNTHETIC PRIMER SEQIDNO: 118 PK81A1XbaF NT 52SYNTHETIC PRIMER SEQIDNO: 119 K81PmBamF NT 47 SYNTHETIC PRIMER SEQIDNO:120 Pm81SmaR2 NT 41 ARABIDOPSIS THALIANA SEQIDNO: 121AtPK220L292F_with_UTR NT 1309 SYNTHETIC PRIMER SEQIDNO: 122 Bn81F NT 25SYNTHETIC PRIMER SEQIDNO: 123 Bn81R NT 32 SYNTHETIC PRIMER SEQIDNO: 124Bn81RAF1 NT 32 SYNTHETIC PRIMER SEQIDNO: 125 Bn81RAF2 NT 32 SYNTHETICPRIMER SEQIDNO: 126 Bn81RAR1 NT 31 SYNTHETIC PRIMER SEQIDNO: 127Bn81RAR2 NT 37 SYNTHETIC PRIMER SEQIDNO: 128 Bn81F1 NT 28 SYNTHETICPRIMER SEQIDNO: 129 Bn81R1 NT 27 SYNTHETIC PRIMER SEQIDNO: 130 Gm81RAR1NT 27 SYNTHETIC PRIMER SEQIDNO: 131 Gm81RAR2 NT 29 SYNTHETIC PRIMERSEQIDNO: 132 Cn81RAR1 NT 29 SYNTHETIC PRIMER SEQIDNO: 133 Cn81RAR2 NT 28SYNTHETIC PRIMER SEQIDNO: 134 A220SacF NT 41 SYNTHETIC PRIMER SEQIDNO:135 Pm81NheF NT 47 SYNTHETIC PRIMER SEQIDNO: 136 Pm81NheR NT 43SYNTHETIC PRIMER SEQIDNO: 137 Gm81RAF1 NT 29 SYNTHETIC PRIMER SEQIDNO:138 Gm81RAF2 NT 31 SYNTHETIC PRIMER SEQIDNO: 139 Zm81RAF1 NT 31SYNTHETIC PRIMER SEQIDNO: 140 Zm81RAF2 NT 27 SYNTHETIC PRIMER SEQIDNO:141 MiR319XbaF NT 31 SYNTHETIC PRIMER SEQIDNO: 142 MiR319BamR NT 33SYNTHETIC PRIMER SEQIDNO: 143 MiPK220F1 NT 40 SYNTHETIC PRIMER SEQIDNO:144 MiPK220R1 NT 35 SYNTHETIC PRIMER SEQIDNO: 145 MiPK220F2 NT 35SYNTHETIC PRIMER SEQIDNO: 146 MiPK220R2 NT 42 ARTIFICIAL SEQUENCESEQIDNO: 147 Synthesized_gene_fragment NT 21 ARABIDOPSIS THALIANASEQIDNO: 148 At4g23713_w_genomic NT 399 ARTIFICIAL SEQUENCE SEQIDNO: 149Artificial_micro_RNA_construct NT 399 ARABIDOPSIS THALIANA SEQIDNO: 150Promoter At2g44790 NT 1475 SYNTHETIC PRIMER SEQIDNO: 151 P790-H3-F NT 31SYNTHETIC PRIMER SEQIDNO: 152 P790-Xb-R NT 31 BRASSICA NAPUS SEQIDNO:153 BnPK220 NT 338 SYNTHETIC PRIMER SEQIDNO: 154 Bn340BamF NT 38SYNTHETIC PRIMER SEQIDNO: 155 Bn340XbaR NT 37 SYNTHETIC PRIMER SEQIDNO:156 Bn340SacF NT 38 SYNTHETIC PRIMER SEQIDNO: 157 Bn340SacR NT 37SYNTHETIC PRIMER SEQIDNO: 158 bWET XbaI F NT 24 SYNTHETIC PRIMERSEQIDNO: 159 bWET BamHI R NT 28 SYNTHETIC PRIMER SEQIDNO: 160 bWET ClaIR NT 28 BRACHYPODIUM SEQIDNO: 161 BdPK220 NT 272 DISTACHYON SYNTHETICPRIMER SEQIDNO: 162 bWx BamHI F NT 31 SYNTHETIC PRIMER SEQIDNO: 163 bWxClaI R NT 30 BRACHYPODIUM SEQIDNO: 164 BdWx intron 1 NT 1174 DISTACHYONSYNTHETIC PRIMER SEQIDNO: 165 bWET BamHI end2 NT 22 SYNTHETIC PRIMERSEQIDNO: 166 BdUBQ PvuI F NT 30 SYNTHETIC PRIMER SEQIDNO: 167 BdUBQTPacI R NT 28 PANICUM VIRGATUM SEQIDNO: 168 Pv(251)PK220 NT 251 SYNTHETICPRIMER SEQIDNO: 169 PvWET XbaI F NT 27 SYNTHETIC PRIMER SEQIDNO: 170PvWET BamHI R NT 27 SYNTHETIC PRIMER SEQIDNO: 171 PvWET ClaI R NT 27SYNTHETIC PRIMER SEQIDNO: 172 PvWET BamHI end1 NT 25 SYNTHETIC PRIMERSEQIDNO: 173 PvWET BamHI end2 NT 21 SORGHUN BICOLOR SEQIDNO: 174Sb(261)PK220 NT 261 SYNTHETIC PRIMER SEQIDNO: 175 SbWET XbaI F NT 26SYNTHETIC PRIMER SEQIDNO: 176 SbWET BamHI R NT 28 SYNTHETIC PRIMERSEQIDNO: 177 SbWET ClaI R NT 28 SORGHUN BICOLOR SEQIDNO: 178 SbWx intron1 NT 273 SYNTHETIC PRIMER SEQIDNO: 179 SbWx BamHI NT 30 SYNTHETIC PRIMERSEQIDNO: 180 SbWx ClaI R NT 34 SYNTHETIC PRIMER SEQIDNO: 181 SbWET BamHIend1 NT 24 SYNTHETIC PRIMER SEQIDNO: 182 SbWET BamHI end2 NT 21 SORGHUNBICOLOR SEQIDNO: 183 SbGOS2 promoter NT 1000 SYNTHETIC PRIMER SEQIDNO:184 SbGOS2 HindIII F NT 28 SYNTHETIC PRIMER SEQIDNO: 185 SbGOS2 HindIIIR NT 30 SORGHUN BICOLOR SEQIDNO: 186 SbUBQ promoter NT 1000 SYNTHETICPRIMER SEQIDNO: 187 SbUBQ PstI F NT 26 SYNTHETIC PRIMER SEQIDNO: 188SbUBQ PstI R NT 28 SORGHUN BICOLOR SEQIDNO: 189 SbUBQ terminator NT 239SYNTHETIC PRIMER SEQIDNO: 190 SbUBQT KpnI F NT 27 SYNTHETIC PRIMERSEQIDNO: 191 SbUBQT KpnI R NT 27 SYNTHETIC PRIMER SEQIDNO: 192 SbUBQPvuI F NT 34 BRASSICA NAPUS SEQIDNO: 193 BnPK220 NT 1302 BRASSICA NAPUSSEQIDNO: 194 BnPK220 AA 433 SYNTHETIC PRIMER SEQIDNO: 195 Bd81RAR1 NT 31SYNTHETIC PRIMER SEQIDNO: 196 Bd81RAR2 NT 32 BRACHYPODIUM SEQIDNO: 197BdPK220 AA 461 DISTACHYON SYNTHETIC PRIMER SEQIDNO: 198 A200A1AgeF NT 53SYNTHETIC PRIMER SEQIDNO: 199 A220AgeR NT 39 SYNTHETIC PRIMER SEQIDNO:200 bWET BamHI end1 NT 18

Sequences

>SEQIDNO: 1 ATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCAGTCTCTAATTCTTTTGATGATCTTCATCACTGTCTCTGCTTCTTCTGCTTCAAATCCTTCTTTAGCTCCTGTTTACTCTTCCATGGCTACATTCTCTCCTCGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTCTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGA >SEQIDNO: 2MRELLLLLLLHFQSLILLMIFITVSASSASNPSLAPVYSSMATFSPRIQMGSGEEDRFDAHKKLLIGLIISFSSLGLIILFCFGFWVYRKNQSPKSINNSDSESGNSFSLLMRRLGSIKTQRRTSIQKGYVQFFDIKTLEKATGGFKESSVIGQGGFGCVYKGCLDNNVKAAVKKIENVSQEAKREFQNEVDLLSKIHHSNVISLLGSASEINSSFIVYELMEKGSLDEQLHGPSRGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGLAVSLDEHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLTPAQCQSLVTWAMPQLTDRSKLPNIVDAVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 3ATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCAGTCTCTAATTCTTTTGATGATCTTCATCACTGTCTCTGCTTCTTCTGCTTCAAATCCTTCTTTAGCTCCTGTTTACTCTTCCATGGCTACATTCTCTCCTCGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTTTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGA >SEQIDNO: 4MRELLLLLLLHFQSLILLMIFITVSASSASNPSLAPVYSSMATFSPRIQMGSGEEDRFDAHKKLLIGLIISFSSLGLIILFCFGFWVYRKNQSPKSINNSDSESGNSFSLLMRRLGSIKTQRRTSIQKGYVQFFDIKTLEKATGGFKESSVIGQGGFGCVYKGCLDNNVKAAVKKIENVSQEAKREFQNEVDLLSKIHHSNVISLLGSASEINSSFIVYELMEKGSLDEQLHGPSRGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGFAVSLDEHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLTPAQCQSLVTWAMPQLTDRSKLPNIVDAVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 5ATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTTTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACAGA >SEQIDNO: 6MGSGEEDRFDAHKKLLIGLIISFSSLGLIILFCFGFWVYRKNQSPKSINNSDSESGNSFSLLMRRLGSIKTQRRTSIQKGYVQFFDIKTLEKATGGFKESSVIGQGGFGCVYKGCLDNNVKAAVKKIENVSQEAKREFQNEVDLLSKIHHSNVISLLGSASEINSSFIVYELMEKGSLDEQLHGPSRGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGFAVSLDEHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLTPAQCQSLVTWAMPQLTDRSKLPNIVDAVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 7ATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTCTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACAGA >SEQIDNO: 8MGSGEEDRFDAHKKLLIGLIISFSSLGLIILFCFGFWVYRKNQSPKSINNSDSESGNSFSLLMRRLGSIKTQRRTSIQKGYVQFFDIKTLEKATGGFKESSVIGQGGFGCVYKGCLDNNVKAAVKKIENVSQEAKREFQNEVDLLSKIHHSNVISLLGSASEINSSFIVYELMEKGSLDEQLHGPSRGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGLAVSLDEHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLTPAQCQSLVTWAMPQLTDRSKLPNIVDAVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 9ATCAAAAACTTTTCTTTTCTTAGCAAAAAAAACAAAAAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCAGTCTCTAATTCTTTTGATGATCTTCATCACTGTCTCTGCTTCTTCTGCTTCAAATCCTTCTTTAGCTCCTGTTTACTCTTCCATGGCTACATTCTCTCCTCGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTCTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACAGAAACACGCCAAAAGAAATCCAAAGCCATTTAGATGATTTTCTTTTATCCTTTGCCTTTATATTTTTTTGTATAGGGTTATGATCCACTCATCTGAAAGTTTGGGGGTAAGAATGTGAGAATATAAGTTTTCAGGGTTGTTGAGTTCTATATAATTATATTTGTTTCTTTTTATTGTCAAATATAATTATATTTTTGT >SEQIDNO: 10AAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCAGTCTCTAATTCTTTTGATGATCTTCATCACTGTCTCTGCTTCTTCTGCTTCAAATCCTTCTTTAGCTCCTGTTTACTCTTCCATGGCTACATTCTCTCCTCGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTCTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACAG >SEQIDNO: 11TCTGTGTCAGGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTCTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACA G >SEQIDNO: 12TCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAA GACCCTC >SEQIDNO: 13TCTGTGTCAGGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTC >SEQIDNO: 14TGTTAAAAGCGATTTATAATTTACACCGTTTTGGTGTATATTTCTATCTATCCTTTTACAAGACCTATATATGTTATGTTATGGTGGTGTACTATTTTAAGTGAGCGACATAGTATTTTCTTCATATAGCTAATTAATCAACAACAATTTCCCAACTTACAACTATTTGCGTACTTTAAACTTATATTGAAAGAGAACTACAAAATTATTTTTTTGTACAAGAGAATTATGGTCTTCGGATCAATAATTTCTCTAGATATAATATGTAAAGCCAACCCTATAATTTGTAAAATCCATGATTTGATATAATTTTCTTTTAAAATTGTGAATTGGCAGACAAAAACAACATTACATTTTGATTTAAATTCATAACTTTGACTTGCTAAGGAAACACCATGATTCATTTTTTGTCATTTGTTACATCATCACTAGAAATATTTGATCTAACTTTATTATGATAATAGACTACATACTACATATGCAGTTACGATTTTAAATACTACATATTTAAGCGTGTTTAAACTGTAACCATATCATATAAAATGACATATCTAAAAGTGATTTTCAATATTTTGATATGATATGTGTTGTAGCACGGATAATGATCTAATTTTTAAGTAATAAGCTTGTTCATTACAAAAGAGAAGAAAGTAGTATTGGGCCATGATTATGTAAGGACAAAATAGGAAGATGTGGAAGAAGCCATTCGAGGGTTTTATTACAAAAACAGAGTATATAATTGGTCATAATGTTTTATTCACTTAATTTAACATTATTGCATTATATTTTCATGAACACATATTTCTTTAACTAAAAATATACACATATTTCTTATTGTAGATGAAGTGAAAAGAACAATATTTGGGTTCACATCTATGGGTGAATCCTTTTAATCACCCCCTAAAATAAAAAAGGTGCCATATTTCTATTTTTAGAGAAAGATATAGAGCACCATTGGAGTGGTTTTGCTCCAAATATAGAGTTTAGAGAAATATATAATACACCATTGGAGATGCTCTAAAATGAATTTATTTATTTATTTAGATGGAAGATTCTAATTGGTTAGAAAAAGAGGAAGTGAATAATAGGATTCACCTATAAGAGTGAACCCAAGTATTTTTAAGAGATAATGTGTAAAGTAAATAGATGGTCATTGTGTGAATTATGAATAGAACCATGGTTTTCCATTTTTAATTGCTTAACATAGGGTAATCAACAATGGGGTTTAATATGTCAATAGACAATAGTAAAGAAAGTATTTGATCTATCCCAAATCTTTCTTCGTTCGTTAGTTCATCACTTTCTTTCTTTTTGGTTATATTAATGGTAGAGAACTAAAAATTCAACTTTTTATTCAAAAGCTCCCTTTCTCTTTCCCTCCTTTATTTGCCATAAAAGTGATTTCAAGAAGACAGCGAGAGAGAAAGTGATAGTTCGTTCACTCTTCGCTTTCTCAAGAATTTCAAAACACCAAAAAAGTCTTTAGATTGAATTTCATCAAAAACTTTTC >SEQIDNO: 15AGACAAGAAAAAAGGAAACAAAATTTTATGAAAGAGATCTCCATTAGAGAAAGAGAGAGCGAGAGAGAGATTAATCTTGGAAGAGCAATCTCACATTCTCACACTGCTCTTAGAAAATCTCTCTTTCACCATTAAAAATCCCAAAGAGTCTGGAGAA >SEQIDNO: 16ATGGGAAAGATTCTTCATCTTCTTCTTCTTCTTCTTAAGGTCTCTGTTCTTGAATTCATCATTAGTGTTTCTGCTTTTACTTCACCTGCTTCACAGCCTTCTCTTTCTCCTGTTTACACTTCCATGGCTTCCTTTTCTCCAGGGATCCACATGGGCAAAGGCCAAGAACACAAGTTAGATGCACACAAGAAACTTCTAATCGCTCTCATAATCACCTCATCTTCTCTAGGACTAATACTTGTATCTTGTTTATGCTTTTGGGTTTATTGGTCTAAGAAATCTCCCAAAAACACCAAGAACTCAGGTGAGAGTAGGATTTCATTATCCAAGAAGGGCTTTGTGCAGTCCTTCGATTACAAGACACTAGAGAAAGCAACAGGCGGTTTCAAAGACGGTAATCTTATAGGACGAGGCGGGTTCGGAGATGTTTACAAGGCCTGTTTAGGCAACAACACTCTAGCAGCAGTCAAAAAGATCGAAAACGTTAGTCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGATTTGTTGAGCAAGATTCACCACCCGAACATCATCTCATTGTTTGGATATGGAAATGAACTCAGTTCGAGTTTTATCGTCTACGAGCTGATGGAAAGCGGATCATTGGATACACAGTTACACGGACCTTCTCGGGGATCGGCTTTAACATGGCACATGCGGATGAAGATTGCTCTTGATACAGCAAGAGCTGTTGAGTATCTCCACGAGCGTTGTCGTCCTCCGGTTATCCACAGAGATCTTAAATCGTCAAATATTCTCCTTGATTCTTCCTTCAACGCCAAGATTTCGGATTTTGGTCTTGCGGTAATGGTGGGGGCTCACGGCAAAAACAACATTAAACTATCAGGAACACTTGGTTATGTTGCTCCAGAATATCTCCTAGATGGAAAATTGACGGATAAGAGTGATGTTTATGCGTTTGGTGTGGTTTTACTTGAACTCTTGTTAGGAAGACGGCCGGTTGAGAAATTGAGTTCGGTTCAGTGTCAATCTCTTGTCACTTGGGCAATGCCCCAACTTACGGATAGATCAAAGCTTCCGAAAATCGTGGATCCGGTTATCAAAGATACAATGGATCATAAGCACTTATACCAGGTGGCAGCCGTGGCAGTGCTTTGTGTACAACCAGAACCGAGTTATCGACCGTTGATAACCGATGTTCTTCACTCACTAGTTCCATTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAATACCATCATCGTCTTGA >SEQIDNO: 17MGKILHLLLLLLKVSVLEFIISVSAFTSPASQPSLSPVYTSMASFSPGIHMGKGQEHKLDAHKKLLIALIITSSSLGLILVSCLCFWVYWSKKSPKNTKNSGESRISLSKKGFVQSFDYKTLEKATGGFKDGNLIGRGGFGDVYKACLGNNTLAAVKKIENVSQEAKREFQNEVDLLSKIHHPNIISLFGYGNELSSSFIVYELMESGSLDTQLHGPSRGSALTWHMRMKIALDTARAVEYLHERCRPPVIHRDLKSSNILLDSSFNAKISDFGLAVMVGAHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLSSVQCQSLVTWAMPQLTDRSKLPKIVDPVIKDTMDHKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLIPSSS >SEQIDNO: 18ATGAAGCAAATTGTTATAACAGCTCTTGTTTTACTACAAGCTTATGTTCTTCATCAATCCACATGTGTTATGTCCCTTACTACACAAGAATCTCCTTCTCCTCAACCTTCTGCTTTCACTCCCGCCTTATCTCCTGATTATCAACAGAGAGAGAAGGAATTGCATAAACAAGAGAGTAACAACATGAGACTGGTTATTTCACTAGCAGCTACATTTTCCTTAGTTGGTATAATCTTACTTTGCTCTCTGCTTTATTGGTTTTGCCATAGGAGAAGAAACCTCAAGAGCTCAGGTTGTGGGTGTAGTGGAATCACATTCTTGAATCGGTTTAGTCGCTCAAAAACATTAGACAAGAGAACTACAAAGCAGGGAACAGTGTCATTGATCGATTACAATATACTAGAAGAAGGAACTAGTGGTTTCAAGGAGAGTAACATTTTGGGTCAAGGTGGATTTGGATGTGTATATTCTGCCACATTAGAGAACAACATTTCAGCTGCGGTTAAGAAGCTAGACTGTGCCAATGAAGATGCAGCAAAGGAATTTAAGAGTGAGGTTGAGATATTGAGTAAGCTCCAGCACCCGAATATAATATCCCTTTTGGGTTATAGCACGAATGATACTGCGAGATTCATTGTCTATGAGCTGATGCCAAACGTTTCTCTGGAATCTCATTTACACGGATCTTCTCAGGGTTCGGCGATCACATGGCCTATGAGGATGAAGATTGCTCTTGATGTAACAAGGGGATTAGAATATTTGCATGAACATTGTCATCCAGCAATCATTCACAGGGACTTGAAATCATCCAACATCTTATTAGATAGCAATTTCAATGCTAAGATTTCAGATTTTGGTCTAGCTGTTGTTGATGGGCCAAAGAACAAGAACCATAAACTTTCCGGGACAGTTGGCTACGTTGCACCAGAGTATCTTCTCAACGGCCAATTGACAGAAAAGAGCGACGTGTATGCTTTTGGAGTAGTGTTATTAGAGCTTTTACTCGGGAAAAAACCTGTGGAGAAACTAGCTCCCGGTGAATGCCAATCCATCATCACTTGGGCAATGCCTTATCTCACTGATAGAACCAAGTTACCAAGCGTCATAGATCCTGCGATTAAAGATACGATGGACTTGAAACACCTTTACCAGGTAGCGGCAGTGGCGATTTTGTGCGTGCAGCCAGAACCGAGTTATAGACCGTTGATTACAGACGTCTTGCATTCTCTTATACCTTTGGTTCCAATGGAACTTGGTGGAACCTTAAAAACCATCAAATGTGCTTCAATGGATCACTGTTAA >SEQIDNO: 19MKQIVITALVLLQAYVLHQSTCVMSLTTQESPSPQPSAFTPALSPDYQQREKELHKQESNNMRLVISLAATFSLVGIILLCSLLYWFCHRRRNLKSSGCGCSGITFLNRFSRSKTLDKRTTKQGTVSLIDYNILEEGTSGFKESNILGQGGFGCVYSATLENNISAAVKKLDCANEDAAKEFKSEVEILSKLQHPNIISLLGYSTNDTARFIVYELMPNVSLESHLHGSSQGSAITWPMRMKIALDVTRGLEYLHEHCHPAIIHRDLKSSNILLDSNFNAKISDFGLAVVDGPKNKNHKLSGTVGYVAPEYLLNGQLTEKSDVYAFGVVLLELLLGKKPVEKLAPGECQSIITWAMPYLTDRTKLPSVIDPAIKDTMDLKHLYQVAAVAILCVQPEPSYRPLITDVLHSLIPLVPMELGGTLKTIKCASMDHC >SEQIDNO: 20ATGAAGACTATGTCCAAATCGTCTTTGCGTTTGCATTTTCTCTCGCTACTCTTACTTTGTTGTGTCTCCCCTTCAAGCTTTGTCATTATAAGATTCATTACACATAATCATTTTGATGGTCTAGTACGTTGTCATCCCCACAAGTTTCAAGCCCTTACGCAGTTCAAGAACGAGTTTGATACCCGCCGTTGCAACCACAGTAACTACTTTAATGGAATCTGGTGTGATAACTCCAAGGTGCGGTCACAAAGCTACGACTACGGGACTGTCTCAGTGGAACTCTCAAATCAAACAGTAGCCTCTTCCAGTTTCATCATCTTCGCTACCTTGATCTCTCTCACAACAACTTCACCTCCTCTTCCCTCCCTTCCGAGTTTGTTTCCCACTTTGCGGAATCTAACCAAGCTCACAGTTTTAGACCTTTCTCATAATCACTTCTCCGGAACTTTGAAGCCCAACAATAGCCTCTTTGAGTTACACCACCTTCGTTACCTTAATCTCGAGGTCAACAACTTCAGTTCCTCACTCCCTTCCGAGTTTGGCTATCTCAACAATTTACAGCACTGTGGCCTCAAAGAGTTCCCAAACATATTCAAGACCCTTAAAAAAATGGAGGCTATAGACGTATCCAACAATAGAATCAACGGGAAAATCCCTGAGTGGTTATGGAGCCTTCCTCTTCTTCATTTAGTGAATATTTTAAATAATTCTTTTGACGGTTTCGAAGGATCAACGGAAGTTTTAGTAAATTCATCGGTTCGGATATTACTTTTGGAGTCAAACAACTTTGAAGGAGCACTTCCTAGTCTACCACACTCTATCAACGCCTTCTCCGCGGGTCATAACAATTTCACTGGAGAGATACCTCTTTCAATCTGCACCAGAACCTCACTTGGTGTCCTTGATCTAAACTACAACAACCTCATTGGTCCGGTTTCTCAATGTTTGAGTAATGTCACGTTTGTAAATCTCCGGAAAAACAATTTGGAAGGAACTATTCCTGAGACTTTCATTGTCGGTTCCTCGATAAGGACACTTGATGTTGGATACAATCGACTAACGGGAAAGCTTCCAAGGTCTCTTTTGAACTGCTCATCTCTAGAGTTTCTAAGCGTTGACAACAACAGAATCAAAGACACATTTCCTTTCTGGCTCAAGGCTTTACCAAAGTTACAAGTCCTTACCCTAAGTTCAAACAAGTTTTATGGTCCTATATCTCCTCCTCATCAAGGTCCTCTCGGGTTTCCAGAGCTGAGAATACTTGAGATATCTGATAATAAGTTTACTGGAAGCTTGTCGTCAAGATACTTTGAGAATTGGAAAGCATCGTCCGCCATGATGAATGAATATGTGGGTTTATATATGGTTTACGAGAAGAATCCTTATGGTGTAGTTGTCTATACCTTTTTGGATCGTATAGATTTGAAATACAAAGGTCTAAACATGGAGCAAGCGAGGGTTCTCACTTCCTACAGCGCCATTGATTTTTCTAGAAATCTACTTGAAGGAAATATTCCTGAATCCATTGGACTTTTAAAGGCATTGATTGCACTAAACTTATCGAACAACGCTTTTACAGGCCATATTCCTCAGTCTTTGGCAAATCTTAAGGAGCTCCAGTCACTAGACATGTCTAGGAACCAACTCTCAGGGACTATTCCTAATGGACTCAAGCAACTCTCGTTTTTGGCTTACATAAGTGTGTCTCATAACCAACTCAAGGGTGAAATACCACAAGGAACACAAATTACTGGGCAATTGAAATCTTCCTTTGAAGGGAATGTAGGACTTTGTGGTCTTCCTCTCGAGGAAAGGTGCTTCGACAATAGTGCATCTCCAACGCAGCACCACAAGCAAGACGAAGAAGAAGAAGAAGAACAAGTGTTACACTGGAAAGCGGTGGCAATGGGGTATGGACCTGGATTGTTGGTTGGATTTGCAATTGCATATGTCATTGCTTCATACAAGCCGGAGTGGCTAACCAAGATAATTGGTCCGAATAAGCGCAG AAACTAG >SEQIDNO: 21MKTMSKSSLRLHFLSLLLLCCVSPSSFVIIRFITHNHFDGLVRCHPHKFQALTQFKNEFDTRRCNHSNYFNGIWCDNSKVRSQSYDYGTVSVELSNQTVASSSFIIFATLISLTTTSPPLPSLPSLFPTLRNLTKLTVLDLSHNHFSGTLKPNNSLFELHHLRYLNLEVNNFSSSLPSEFGYLNNLQHCGLKEFPNIFKTLKKMEAIDVSNNRINGKIPEWLWSLPLLHLVNILNNSFDGFEGSTEVLVNSSVRILLLESNNFEGALPSLPHSINAFSAGHNNFTGEIPLSICTRTSLGVLDLNYNNLIGPVSQCLSNVTFVNLRKNNLEGTIPETFIVGSSIRTLDVGYNRLTGKLPRSLLNCSSLEFLSVDNNRIKDTFPFWLKALPKLQVLTLSSNKFYGPISPPHQGPLGFPELRILEISDNKFTGSLSSRYFENWKASSAMMNEYVGLYMVYEKNPYGVVVYTFLDRIDLKYKGLNMEQARVLTSYSAIDFSRNLLEGNIPESIGLLKALIALNLSNNAFTGHIPQSLANLKELQSLDMSRNQLSGTIPNGLKQLSFLAYISVSHNQLKGEIPQGTQITGQLKSSFEGNVGLCGLPLEERCFDNSASPTQHHKQDEEEEEEQVLHWKAVAMGYGPGLLVGFAIAYVIASYKPEWLTKIIGPNKRRN >SEQIDNO: 22ATGACTTCCTCTCGCCGTCTTCTTCTTCCTCTCGGAGCATCGCTCACTAGAGGAAGATTTTCTTCCGATCAAATCCGAAATGGATTTCTAAGAAACTTCCGTGGATTCGCCACCGTAACTTCGTCGGAACCGGCCTTAGCCAATCTGGAAGCGAAATATGCCGTAGCGTTGCCAGAATGTTCAACAGTAGAGGACGAGATCACGAAGATCCGTCATGAATTCGAGTTAGCGAAACAGAGGTTTCTTAATATCCCTGAAGCTATTAATAGTATGCCGAAGATGAATCCTCAAGGGATATATGTGAATAAGAATCTGAGATTGGATAATATACAAGTTTATGGATTTGATTATGATTACACTTTGGCACATTACTCTTCTCACTTACAGAGTTTGATCTATGATCTTGCCAAGAAACATATGGTTAATGAGTTTAGATATCCTGATGTTTGCACTCAGTTTGAGTATGATCCTACTTTTCCAATCCGTGGGTTGTACTATGATAAACTAAAAGGATGCCTCATGAAATTGGATTTCTTCGGTTCAATCGAGCCAGATGGGTGTTATTTTGGTCGTCGTAAGCTTAGTAGGAAGGAAATAGAAAGCATGTATGGAACGCGGCACATAGGTCGTGATCAAGCGAGAGGTTTGGTGGGATTGATGGATTTCTTCTGTTTTAGCGAGGCGTGTCTTATAGCAGACATGGTGCAATATTTTGTTGACGCCAAACTTGAGTTTGATGCCTCTAACATCTACAATGATGTCAATCGTGCTATTCAACATGTCCATAGAAGTGGATTGGTTCATAGAGGAATTCTTGCTGATCCCAACAGATATTTGCTAAAAAATGGTCAGCTTCTACGTTTCCTGAGAATGCTAAAAGATAAAGGAAAGAAGCTTTTTTTGCTGACCAACTCTCCGTATAATTTTGTTGATGGCGGAATGCGCTTTCTAATGGAGGAATCTTTTGGCTTCGGAGATTCCTGGCGAGAACTCTTTGATGTTGTGATTGCTAAAGCAAATAAACCAGAATTTTACACATCTGAGCACCCTTTCCGTTGTTATGATTCGGAGAGGGATAATTTGGCATTTACAAAAGTGGATGCATTTGACCCAAAGAAAGTTTATTATCATGGTTGTCTTAAATCCTTCCTTGAAATCACAAAGTGGCATGGCCCTGAGGTGATTTATTTCGGAGATCACTTATTTAGTGATCTAAGAGGGCCTTCAAAAGCTGGTTGGCGAACTGCTGCCATAATTCATGAGCTCGAGCGAGAGATACAGATACAAAATGATGATAGCTACCGGTTTGAGCAGGCCAAGTTCCATATTATCCAAGAGTTACTCGGTAGATTTCACGCGACTGTATCAAACAATCAGAGAAGTGAAGCATGCCAATCACTTTTGGATGAGCTGAACAATGCGAGGCAGAGAGCAAGAGACACGATGAAACAAATGTTCAACAGATCGTTTGGAGCTACATTTGTCACAGACACTGGTCAAGAATCAGCATTCTCTTATCACATCCACCAATACGCAGACGTTTATACCAGTAAACCTGAGAACTTTCTGTTATACCGACCTGAAGCCTGGCTTCACGTTCCTTACGATATCAAGATCATGCCACATCATGTCAAGGTTGCTTCAACCCTTTTCAAAACCTGA >SEQIDNO: 23MTSSRRLLLPLGASLTRGRFSSDQIRNGFLRNFRGFATVTSSEPALANLEAKYAVALPECSTVEDEITKIRHEFELAKQRFLNIPEAINSMPKMNPQGIYVNKNLRLDNIQVYGFDYDYTLAHYSSHLQSLIYDLAKKHMVNEFRYPDVCTQFEYDPTFPIRGLYYDKLKGCLMKLDFFGSIEPDGCYFGRRKLSRKEIESMYGTRHIGRDQARGLVGLMDFFCFSEACLIADMVQYFVDAKLEFDASNIYNDVNRAIQHVHRSGLVHRGILADPNRYLLKNGQLLRFLRMLKDKGKKLFLLTNSPYNFVDGGMRFLMEESFGFGDSWRELFDVVIAKANKPEFYTSEHPFRCYDSERDNLAFTKVDAFDPKKVYYHGCLKSFLEITKWHGPEVIYFGDHLFSDLRGPSKAGWRTAAIIHELEREIQIQNDDSYRFEQAKFHIIQELLGRFHATVSNNQRSEACQSLLDELNNARQRARDTMKQMFNRSFGATFVTDTGQESAFSYHIHQYADVYTSKPENFLLYRPEAWLHVPYDIKIMPHHVKVASTLFKT >SEQIDNO: 24ATGGAGATTCCGGCGGCGCCGCCGCCTCCATTGCCGGTGCTGTGCTCGTACGTCGTCTTCTTGCTGCTGCTGTCTTCGTGCTCACTGGCCAGAGGGAGGATCGCGGTTTCTTCCCCGGGCCCGTCGCCTGTGGCCGCCGCCGTTACAGCCAATGAGACCGCTTCATCCTCTTCTTCTCCGGTGTTTCCGGCCGCTCCTCCCGTCGTGATCACAGTGGTGAGGCACCACCATTACCACCGGGAGCTGGTCATCTCCGCTGTCCTCGCCTGCGTCGCCACCGCCATGATCCTCCTCTCCACACTCTACGCCTGGACGATGTGGCGGCGGTCTCGCCGGACCCCCCACGGCGGCAAGGGCCGCGGCCGGAGATCAGGGATCACACTGGTGCCAATCCTGAGCAAGTTCAATTCAGTGAAGATGAGCAGGAAGGGGGGCCTTGTGACGATGATCGAGTACCCGTCGCTGGAGGCGGCGACAGGCAAGTTCGGCGAGAGCAATGTGCTCGGTGTCGGCGGCTTCGGTTGCGTTTATAAGGCGGCGTTTGATGGCGGTGCCACCGCCGCCGTGAAGAGGCTTGAAGGCGGCGGGCCGGATTGCGAGAAGGAATTCGAGAATGAGCTGGATTTGCTTGGCAGGATCAGGCACCCAAACATAGTGTCTCTCCTGGGCTTCTGTGTCCATGGTGGCAATCACTACATTGTTTATGAGCTCATGGAGAAGGGATCATTGGAGACACAGCTGCATGGGTCTTCACATGGATCTGCTCTGAGCTGGCACGTTCGGATGAAGATCGCGCTCGATACGGCGAGGGGATTAGAGTATCTTCATGAGCACTGCAATCCACCTGTGATCCATAGGGATCTGAAACCTTCTAATATACTTTTAGATTCAGACTTCAATGCTAAGATTGCAGATTTTGGCCTTGCGGTCACCGGTGGGAATCTCAACAAAGGGAACCTGAAGCTTTCCGGGACCTTGGGTTATGTAGCCCCTGAGTACTTATTAGATGGGAAGTTGACTGAGAAGAGCGATGTATACGCATTTGGAGTAGTGCTTCTAGAGCTCCTGATGGGAAGGAAGCCTGTTGAGAAAATGTCACCATCTCAGTGCCAATCAATTGTGTCATGGGCTATGCCTCAGCTGACCGACAGATCGAAGCTCCCCAACATAATTGACCTGGTGATCAAGGACACCATGGACCCAAAACACTTGTACCAAGTTGCAGCAGTGGCTGTTCTATGTGTGCAGCCCGAACCGAGCTACAGACCACTGATAACAGATGTTCTCCACTCTCTTGTTCCTCTAGTGCCTGCGGAGCTCGGAGGAACACTCAGGGTTGCAGAGCCACCTTCACCTTCTCCAGACCAAAGACATTATCCTTGTTGA >SEQIDNO: 25ATGAAGAAACTGGTTCATCTTCAGTTTTTGTTTCTTGTCAAGATCTTTGCTACTCAATTCCTCACTCCTTCTTCATCATCTTTTGCTGCTTCAAATCCTTCTATAGCTCCTGTTTACACCTCCATGACTACTTTCTCTCCAGGAATTCAAATGGGAAGTGGTGAAGAACACAGATTAGATGCACATAAGAAACTCCTGATTGGTCTTATAATCAGTTCCTCTTCTCTTGGTATCATAATCTTGATTTGCTTTGGCTTCTGGATGTACTGTCGCAAGAAAGCTCCCAAACCCATCAAGATTCCGGATGCCGAGAGTGGGACTTCATCATTTTCAATGTTTGTGAGGCGGCTAAGCTCAATTAAAACTCACAGAACATCTAGCAATCAGGGTTATGTGCAGCGTTTCGATTCCAAGACGCTAGAGAAAGCGACAGGCGGTTTCAAAGACAGTAATGTAATCGGACAGGGCGGTTTCGGATGCGTTTACAAGGCTTCTTTGGACAGCAACACTAAAGCAGCGGTTAAAAAGATCGAAAACGTTACCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGAGCTGTTGAGCAAGATCCAGCACTCCAATATTATATCATTGTTGGGCTCTGCAAGTGAAATCAACTCGAGTTTCGTCGTTTATGAGTTGATGGAGAAAGGATCCTTAGATGATCAGTTACATGGACCTTCGTGTGGATCCGCTCTAACATGGCATATGCGTATGAAGATTGCTCTAGATACAGCTAGAGGACTAGAGTATCTCCATGAACATTGTCGTCCACCAGTTATCCACAGGGACCTGAAATCGTCTAATATTCTTCTTGATTCTTCCTTCAATGCCAAGATTTCAGATTTTGGTCTGGCTGTATCGGTTGGAGTGCATGGGAGTAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATATCTCCTAGACGGAAAGTTGACGGATAAGAGTGATGTCTATGCATTTGGGGTGGTTCTTCTTGAACTTTTGTTGGGTAGGCGGCCGGTTGAGAAATTGAGTCCATCTCAGTGTCAATCTCTTGTGACTTGGGCAATGCCACAACTTACCGATAGATCGAAACTCCCAAACATCGTGGATCCGGTTATAAAAGATACAATGGATCTTAAGCACTTATACCAAGTAGCAGCCATGGCTGTGCTGTGCGTACAGCCAGAACCGAGTTACCGGCCGCTGATAACCGATGTTCTTCATTCACTTGTTCCATTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACCCGATGA >SEQIDNO: 26MKKLVHLQFLFLVKIFATQFLTPSSSSFAASNPSIAPVYTSMTTFSPGIQMGSGEEHRLDAHKKLLIGLIISSSSLGIIILICFGFWMYCRKKAPKPIKIPDAESGTSSFSMFVRRLSSIKTHRTSSNQGYVQRFDSKTLEKATGGFKDSNVIGQGGFGCVYKASLDSNTKAAVKKIENVTQEAKREFQNEVELLSKIQHSNIISLLGSASEINSSFVVYELMEKGSLDDQLHGPSCGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGLAVSVGVHGSNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLSPSQCQSLVTWAMPQLTDRSKLPNIVDPVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 27ATTTTTGGTGTTGAAATGATGCACAACGGATCTTTGGAATCCCAATTGCATGGTCCGTCTCATGGAACTGGCTTAAGCTGGCAGCATCGAATGAAAATTGCACTTGATATTGCACGAGGACTAGAGTATCTTCACGAGCGCTGTACCCCGCCTGTGATTCATAGAGATCTGAAATCGTCCAACATTCTTCTAGGTTCGAACTACAATGCTAAACTTTCTGATTTCGGGCTCGCGATTACTGGTGGGATTCAGGGCAAGAACAACGTAAAGCTTTCGGGAACATTAGGTTATGTAGCTCCAGAATACCTCTTAGATGGTAAACTTACTGATAAAAGTGATGTTTATGCGTTTGGAGTTGTACTTCTTGAACTTTTGATAGGTAGAAAACCAGTGGAGAAAATGTCACCATCTCAATGCCAATCTATCGTTACATGGGCAATGCCTCAACTAACCGACCGATCAAAGCTTCCTAACATCGTTGATCCCGTGATTAGAGATACAATGGACTTGAAGCACTTGTATCAAGTTGCTGCGGTTGCTGTGCTATGTGTACAACCGGAACCGAGTTACAGGCCATTGATAACAGATGTTTTGCATTCGTTCATCCCACTTGTACCTGTTGAGCTTGGAGGGTCGCTAAGAGTTACCGAATCTTGA >SEQIDNO: 28IFGVEMMHNGSLESQLHGPSHGTGLSWQHRMKIALDIARGLEYLHERCTPPVIHRDLKSSNILLGSNYNAKLSDFGLAITGGIQGKNNVKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLIGRKPVEKMSPSQCQSIVTWAMPQLTDRSKLPNIVDPVIRDTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSFIPLVPVELGGSLRVTES >SEQIDNO: 29AATTCGGCACGAGGGCTGGATTCCAGTTTTAATGCAAAGCTTTCAGATTTTGGCCTTTCTGTGACTGCTGGAACCCAGAGTAGGAATGTTAAGATCTCTGGAACTCTGGGTTATGTTGCCCCGGAGTACCTATTAGAAGGAAAACTAACTGATAAAAGTGATGTATATGCTTTCGGAGTTGTATTGCTGGAACTTTTGATGGGGAGAAGGCCTGTGGAAAAGATGTCACCAACTCAATGTCAATCAATGGTCACATGGGCCATGCCTCAGCTCACCGATAGATCAAAGCTTCCAAACATTGTGGATCCAGTAATTAGAGACACAATGGATTTAAAGCACTTATACCAGGTAGCCGCTGTGGCAGTGCTATGTATACAACCTGAACCAAGTTATAGGCCATTGATAACCGACGTTCTGCATTCCCTCATTCCTCTTGTACCTACCGACCTTGGAGGGTCACTCCGAGTGACCTAA >SEQIDNO: 30NSARGLDSSFNAKLSDFGLSVTAGTQSRNVKISGTLGYVAPEYLLEGKLTDKSDVYAFGVVLLELLMGRRPVEKMSPTQCQSMVTWAMPQLTDRSKLPNIVDPVIRDTMDLKHLYQVAAVAVLCIQPEPSYRPLITDVLHSLIPLVPTDLGGSLRVT >SEQIDNO: 31GGATTGTGTTTGTGGCTTTATCATTTGAAGTACTCCTTCAAATCCAGTAACAAGAATGCAAAGAGCAAAGATTCTGAGAATGGAGTTGTGTTATCATCATTTTTGGGCAAATTCACTTCTGTGAGGATGGTTAGTAAGAAGGGATCTGCTATTTCATTTATTGAGTATAAGCTGTTAGAGAAAGCCACCGACAGTTTTCATGAGAGTAATATATTGGGTGAGGGTGGATTTGGATGTGTTTACAAGGCTAAATTGGATGATAACTTGCACGTCGCTGTCAAAAAATTAGATTGTGCAACACAAGATGCCGGCAGAGAATTTGAGAATGAGGTGGATTTGCTGAGTAATATTCACCACCCAAATGTTGTTTGTCTGTTGGGTTATAGTGCTCATGATGACACAAGGTTTATTGTTTATGAATTGATGGAAAATCGGTCCCTTGATATTCAATTGCATGGTCCTTCTCATGGATCAGCATTGACTTGGCATATGCGAATGAAAATTGCTCTTGATACCGCTAGAGGATTAGAATATTTACATGAGCACTGCAACCCTGCAGTCATTCATAGAGATCTGAAATCCTCCAATATACTTCTAGATTCCAAGTTTAATGCTAAGCTCTCAGATTTTGGTCTTGCCATAACCGATGGATCCCAAAACAAGAACAATCTTAAGCTTTCGGGCACTTTGGGATATGTGGCTCCCGAGTATCTTTTAGATGGTAAATTGACAGACAAGAGTGATGTCTATGCTTTTGGAGTTGTGCTTCT >SEQIDNO: 32GLCLWLYHLKYSFKSSNKNAKSKDSENGVVLSSFLGKFTSVRMVSKKGSAISFIEYKLLEKATDSFHESNILGEGGFGCVYKAKLDDNLHVAVKKLDCATQDAGREFENEVDLLSNIHHPNVVCLLGYSAHDDTRFIVYELMENRSLDIQLHGPSHGSALTWHMRMKIALDTARGLEYLHEHCNPAVIHRDLKSSNILLDSKFNAKLSDFGLAITDGSQNKNNLKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLL >SEQIDNO: 33GCATTGACATGGCATCTTAGGATGAAAATTGCCCTTGATGTAGCTAGAGGATTAGAATTTTTGCATGAGCACTGCCACCCAGCAGTGATCCATAGAGATCTGAAATCATCTAATATCCTTCTGGATTCAAATCTCAATGCTAAGCTATCTGATTTTGGTCTTGCCATTCTTGATGGGGCTCAAAATAAGAACAACATCAAGCTTTCTGGAACCTTGGGCTATGTAGCTCCAGAGTACCTCTTAGATGGTAAATTGACTGACAAGAGTGATGTTTATGCTTTTGGAGTGGTGCTTTTGGAGCTTCTCCTGAGAAGAAAGCCTGTGGAGAAGCTGGCACCAGCTCAATGCCAATCTATAGTCACATGGGCTATGCCTCAGCTGACAGATAGATCAAAGCTTCCAAACATCGTGGATCCTGTGATTAGAAATGCTATGGATATAAAGCACTTATTCCAGGTTGCTGCAGTCGCTGTGCTATGCGTGCAGCCTGAACCAAGCTATCGACCACTGATAACAGATGTGTTGCATTCCCTTGTTCCCCTTGTTCCTATGGAGCTTGGCGGGACGCTCAGAGTTGAACGACCTGCTTCTGTGACCTCTCTGTTGATTGATTCTACCTGA >SEQIDNO: 34ALTWHLRMKIALDVARGLEFLHEHCHPAVIHRDLKSSNILLDSNLNAKLSDFGLAILDGAQNKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLRRKPVEKLAPAQCQSIVTWAMPQLTDRSKLPNIVDPVIRNAMDIKHLFQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPMELGGTLRVERPASVTSLLIDST >SEQIDNO: 35ACTGAGGTGACCCGGAAGAAAAACAGGGTAAAGCTATCGGGCACTTTGGGTTATGTAGCCCCAGAATATGTCTTGGATGGTAAATTGACTGATAAGAGTGATGTCTATGCCTTTGGAGTTGTGCTTTTGGAGCTCCTTTTGAGAAGAAGGCCTCTTGAGATAGTAGCACCCACTCAGTGCCAGTCTATTGTTACATGGGCCATGCCTCAGCTGACCGACCGAACTAAGCTTCCAGATATTGTGGATCCTGTAATTAGAGATGCGATGGATGTCAAGCACTTATACCAGGCAGCTGCTGTTGCTGTTTTGTGTCTGCAACCAGAACCGATCTACCGGCCACTGATAACGGATGTACTCCACTCTCTCATTCCACTTGTACCCGTTGAACTTGGGGGAACGCTGAAGACCTAG >SEQIDNO: 36TEVTRKKNRVKLSGTLGYVAPEYVLDGKLTDKSDVYAFGVVLLELLLRRRPLEIVAPTQCQSIVTWAMPQLTDRTKLPDIVDPVIRDAMDVKHLYQAAAVAVLCLQPEPIYRPLITDVLHSLIPLVPVELGGTLKT >SEQIDNO: 37ACGAGGCCTCGTGCCATACTTTTGGATTCAGATTTCAATGCCAAGATTTCGGATTTCGGTCTTGCAGTGTCAAGTGGAAATCGCACCAAAGGTAATCTGAAGCTTTCCGGAACTTTGGGCTATGTTGCTCCTGAGTACTTATTAGACGGGAAGTTGACAGAGAAGAGTGATGTATATGCGTTCGGAGTAGTACTTCTTGAGCTTTTGTTAGGAAGGAGGCCAATTGAGAAGATGGCCCCATCTCAATGCCAATCAATTGTTACATGGGCCATGCCTCAGCTAATTGACAGATCAAAGCTCCCAACCATAATTGACCCCGTGATCAGGAACACGATGGACCTGAAGCACTTGTACCAAGTTGCTGCAGTGGCTGTGCTCTGTGTGCAGCCAGAACCAAGTTATAGGCCACTAATCACAGATGTGCTCCACTCTCTGATTCCCCTGGTGCCCATGGAGCTCGGAGGGTCACTGAGGGCTACCTTGGAATCGCCTCGCGTATCACAACATCGTTCTCCCTGCTGA >SEQIDNO: 38TRPRAILLDSDFNAKISDFGLAVSSGNRTKGNLKLSGTLGYVAPEYLLDGKLTEKSDVYAFGVVLLELLLGRRPIEKMAPSQCQSIVTWAMPQLIDRSKLPTIIDPVIRNTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLIPLVPMELGGSLRATLESPRVSQHRSPC >SEQIDNO: 39CCTTTATTGAATAGATTGAACTCCTTCCGTGGTTCTAGGAGAAAGGGATGTGCATATATAATTGAATATTCTCTGCTGCAAGCAGCCACAAATAATTTTAGTACAAGTGACATCCTTGGAGAGGGTGGTTTTGGGTGTGTATACAGAGCTAGGTTAGATGATGATTTCTTTGCTGCTGTGAAGAAGTTAGATGAGGGCAGCAAGCAGGCTGAGTATGAATTTCAGAATGAAGTTGAACTAATGAGCAAAATCAGACATCCAAATCTTGTTTCTTTGCTGGGGTTCTGCATTCATGGGAAGACTCGGTTGCTAGTCTACGAGCTCATGCAAAATGGTTCTTTGGAAGACCAATTACATGGGCCATCTCATGGATCCGCACTTACATGGTACCTGCGCATGAAAATAGCCCTTGATTCAGCAAGGGGTCTAGAACACTTGCACGAGCACTGCAATCCTGCTGTGATTCATCGTGATTTCAAATCATCAAATATCCTTCTGGATGCAAGCTTCAATGCCAAGCTTTCAGATTTTGGTCTTGCAGTAACAGCTGCAGGAGGTATTGGTAATGCTAATGTCGAGCTACTGGGCACTTTGGGATATGTAGCTCCAGAATACCTGCTTGATGGCAAGTTGACGGAGAAAAGTGATGTCTATGGATTTGGAGTTGTTCTTTTGGAGCTAATTATGGGAAGAAAGCCAGTTGATAAATCTGTGGCAACTGAAAGTCAATCGCTA GTTTC >SEQIDNO: 40PLLNRLNSFRGSRRKGCAYIIEYSLLQAATNNFSTSDILGEGGFGCVYRARLDDDFFAAVKKLDEGSKQAEYEFQNEVELMSKIRHPNLVSLLGFCIHGKTRLLVYELMQNGSLEDQLHGPSHGSALTWYLRMKIALDSARGLEHLHEHCNPAVIHRDFKSSNILLDASFNAKLSDFGLAVTAAGGIGNANVELLGTLGYVAPEYLLDGKLTEKSDVYGFGVVLLELIMGRKPVDKSVATESQSL VS >SEQIDNO: 41ATGAAAATGAAGCTTCTCCTCATGCTTCTTCTTCTTGTTCTTCTTCTTCACCAACCCATTTGGGCTGCAGACCCTCCTGCTTCTTCTCCTGCTTTATCTCCAGGGGAGGAGCAGCATCACCGGAATAATAAAGTGGTAATAGCTATCGTCGTAGCCACCACTGCACTTGCTGCACTCATTTTCAGTTTCTTATGCTTCTGGGTTTATCATCATACCAAGTATCCAACAAAATCCAAATTCAAATCCAAAAATTTTCGAAGTCCAGATGCAGAGAAGGGGATCACCTTAGCACCGTTTGTGAGTAAATTCAGTTCCATCAAGATTGTTGGCATGGACGGGTATGTTCCAATAATTGACTATAAGCAAATAGAAAAAACGACCAATAATTTTCAAGAAAGTAACATCTTGGGTGAGGGCGGTTTTGGACGTGTTTACAAGGCTTGTTTGGATCATAACTTGGATGTTGCAGTCAAAAAACTACATTGTGAGACTCAACATGCTGAGAGAGAATTTGAGAACGAGGTGAATATGTTAAGCAAAATTCAGCATCCGAATATAATATCTTTACTGGGTTGTAGCATGGATGGTTACACGAGGCTCGTTGTCTATGAGCTGATGCATAATGGATCATTGGAAGCTCAGTTACATGGACCTTCTCATGGCTCGGCATTGACTTGGCACATGAGGATGAAGATTGCTCTTGACACAGCAAGAGGATTAGAATATCTGCACGAGCACTGTCACCCTGCAGTGATCCATAGGGATATGAAATCTTCTAATATTCTCTTAGATGCAAACTTCAATGCCAAGCTGTCTGATTTTGGTCTTGCCTTAACTGATGGGTCCCAAAGCAAGAAGAACATTAAACTATCGGGTACCTTGGGATACGTAGCACCGGAGTATCTTCTAGATGGTAAATTAAGTGATAAAAGTGATGTCTATGCTTTTGGGGTTGTGCTATTGGAGCTCCTACTAGGAAGGAAGCCAGTAGAAAAACTGGTACCAGCTCAATGCCAATCTATTGTCACATGGGCCATGCCACACCTCACGGACAGATCCAAGCTTCCAAGCATTGTGGATCCAGTGATTAAGAATACAATGGATCCCAAGCACTTGTACCAGGTTGCTGCTGTAGCTGTGCTGTGCGTGCAACCAGAACCTAGTTACCGTCCACTGATCATTGATGTTCTTCACTCACTCATCCCTCTTGTTCCCATTGAGCTTGGAGGAACACTAAGAGTTTCACAAGTAATT >SEQIDNO: 42MKMKLLLMLLLLVLLLHQPIWAADPPASSPALSPGEEQHHRNNKVVIAIVVATTALAALIFSFLCFWVYHHTKYPTKSKFKSKNFRSPDAEKGITLAPFVSKFSSIKIVGMDGYVPIIDYKQIEKTTNNFQESNILGEGGFGRVYKACLDHNLDVAVKKLHCETQHAEREFENEVNMLSKIQHPNIISLLGCSMDGYTRLVVYELMHNGSLEAQLHGPSHGSALTWHMRMKIALDTARGLEYLHEHCHPAVIHRDMKSSNILLDANFNAKLSDFGLALTDGSQSKKNIKLSGTLGYVAPEYLLDGKLSDKSDVYAFGVVLLELLLGRKPVEKLVPAQCQSIVTWAMPHLTDRSKLPSIVDPVIKNTMDPKHLYQVAAVAVLCVQPEPSYRPLIIDVLHSLIPLVPIELGGTLRVSQVI >SEQIDNO: 43ACTCAAGCATCAAAATATTGTAAATCTTTTGGGTATTGTGTTCATGATGACACAAGGTTTTTGGTCTATGAAATGATGCATCAAGGCTCTTTGGACTCACAATTGCATGGACCAACTCATGGAACCGCATTAACCTGGCATCGAAGAATGAAAGTCGCACTTGATATTGCTCGAGGATTAGAGTATCTTCATGAACGATGCAACCCGCCTGTGATTCATAGAGATCTTAAGTCATCGAACATTTTGCTAGATTCCAATTTCAATGCTAAAATTTCGAATTTTGCACTTGCTACCACTGAGCTCCATGCGAAGAACAAAGTTAAGCTTTCGGCTACTTCTGGTTATTTGGCTCCGGAATACCTATCAGAAGGTAAACTTACCGATAAAAGCGACGTATATGCATTCGGAGTAGTACTTCTTGGGCTTTTAATCGGTAGAAAACCAGTGGAGAAAATGTCACCATCTTTATTTCAATCTATTGTCACATGGGCAATGCCTCAGTTAACAGACCGGTCAAAGCTTCCAAACATCGTTGACCCTGTGATTAGAGATACAATGGACCTGAAGCACTTATATCAAGTTGCTGCTGTAGCCGTACTTTGCGTGCAACCCGAACCAAGTTACAGACCGTTGATTACAGACGTACTACACTCATTCATTCCACTCGTACCCGTTGATCTTGGAGGGTCATTAAGAGCTTAA >SEQIDNO: 44TQASKYCKSFGYCVHDDTRFLVYEMMHQGSLDSQLHGPTHGTALTWHRRMKVALDIARGLEYLHERCNPPVIHRDLKSSNILLDSNFNAKISNFALATTELHAKNKVKLSATSGYLAPEYLSEGKLTDKSDVYAFGVVLLGLLIGRKPVEKMSPSLFQSIVTWAMPQLTDRSKLPNIVDPVIRDTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSFIPLVPVDLGGSLRA >SEQIDNO: 45CGATCATTTCGTTGCGGCTGTAAAAAACTCCATGGTCCAGAACCAGATGCCCAAAAAGGGTTTGAGAATGAAGTAGATTGGTTAGGTAAACTCAAGCATCAAAATATTGTAAATTTTTTGGGTTATTGTGTTCATGATGACACAAGGTTTTTGGTCTATGAAATGATGCATCAAGGCTCTTTGGACTCACAATTGCATGGACCAACTCATGGAACCGCATTAACCTGGCATCGAAGAATGAAAGTCGCACTTGATATTGCTCGAGGATTAGAGTATCTTCATGAACGATGCAACCCGCCTGTGATTCATAGAGATCTCAAGTCATCGAACATTTTGCTAGATTCCAATTTCAATGCTAAAATTTCGAATTTTGCACTTGCTACCACTGAGCTCCATGCGAAGAACAAAGTTAAGCTTTCGGGTACTTCTGGTTATTTGGCTCCGGAATACCTATCCGAAGGTAAACTTACCGATAAAAGTGATGTATATGCATTCGGAGTAGTACTTCTTGAGCTTTTAATCGGTAGAAAACCAGTGGAGAAAATGTCACCATCTTTATTTCAATCTATTGTCACATGGGCAATGCCTCAGCTAACAGACCGGTCAAAGCTTCCAAACATTGTTGACCCTGTGATTAGAGATACAATGGACCTGAAGCACTTGTATCAAGTTGCTGCTGTAGCCGTACTTTGCGTGCAACCCGAACCAAGTTACAGACCGTTGATTACAGACGTACTACACTCATTCATTCC >SEQIDNO: 46RSFRCGCKKLHGPEPDAQKGFENEVDWLGKLKHQNIVNFLGYCVHDDTRFLVYEMMHQGSLDSQLHGPTHGTALTWHRRMKVALDIARGLEYLHERCNPPVIHRDLKSSNILLDSNFNAKISNFALATIELHAKNKVKLSGTSGYLAPEYLSEGKLTDKSDVYAFGVVLLELLIGRKPVEKMSPSLFQSIVTWAMPQLTDRSKLPNIVDPVIRDTMDLKHLYQVAAVAVLCVQPEPSYRPLITDV LHSFIP >SEQIDNO: 47ATGATGCATCAAGACTCTTTGGACTCACAATTGCATGGACCAACTCATGGAACCGCATTAACCTGGCATCGAAGAATGAAAGTCGCACTTGATATTGCTCGAGGATTAGAGTATCTTCATGAACGATGCAACCCGCCTGTGATTCATAGAGATCTCAAGTCATCGAACATTTTGCTAGATTCCAATTTCAATGCTAAAATTTCGAATTTTGCACTTGCTACCACTGAGCTCCATGCGAAGAACAAAGTTAAGCTTTCGGGTACTTCTGGTTATTTGGCTCCGGAATACCTATCCGAAGGTAAACTTACCGATAAAAGTGATGTATATGCATTCGGAGTAGTACTTCTTGAGCTTTTAATCGGTAGAAAACCAGTGGAGAAAATGTCACCATCTTTATTTCAATCTATTGTCACATGGGCAATGCCTCAGCTAACAGACCGGTCAAAGCTTCCAAACATTGTTGACCCTGTGATTAGAGATACAATGGACCTGAAGCACTTGTATCAAGTTGCTGCTGTAGCCGTACTTTGCGTGCAACCCGAACCAAGTTACAGACCGTTGATTACAGACGTACTACACTCATTCATTCCACTCGTACCCGTTGATCTTGGAGGGTCATTAAGAGCTTAA >SEQIDNO: 48MMHQDSLDSQLHGPTHGTALTWHRRMKVALDIARGLEYLHERCNPPVIHRDLKSSNILLDSNFNAKISNFALATIELHAKNKVKLSGTSGYLAPEYLSEGKLTDKSDVYAFGVVLLELLIGRKPVEKMSPSLFQSIVTWAMPQLTDRSKLPNIVDPVIRDTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSFIPLVPVDLGGSLRA >SEQIDNO: 49AATTTGAGAGGTGAGCTGGATTTGCTTCAGAGGATTCAGCATTCGAATATAGTGTCCCTTGTGGGCTTCTGCATTCATGAGGAGAACCGCTTCATTGTTTATGAGCTGATGGTGAATGGATCACTTGAAACACAGCTTCATGGGCCATCACATGGATCAGCTCTGAGTTGGCACATTCGGATGAAGATTGCTCTTGATACAGCAAGGGGATTGGAGTATCTTCACGAGCACTGCAATCCACCAATCATCCATAGGGATCTGAAGTCGTCTAACATACTTTTGAATTCAGACTTTAATGCAAAGATTTCAGATTTTGGCCTTGCAGTGACAAGTGGAAATCGCAGCAAAGGGAATCTGAAGCTTTCCGGTACTTTGGGTTATGTTGCCCCTGAGTACTTACTAGATGGGAAGTTGACTGAGAAGAGCGATGTATATGCATTTGGAGTAGTACTTCTTGAGCTTCTTTTGGGAAGGAGGCCAGTTGAGAAGATGGCACCATCTCAGTGTCAATCAATTGTTACATGGGCCATGCCCCAGCTAATTGACAGATCCAAGCTCCCTACCATAATCGACCCCGTGATCAGGGACACGATGGATCGGAAGCACTTGTACCAAGTTGCTGCAGTGGCTGTGCTCTGCGTGCAGCCAGAACCAAGCTACAGGCCACTGATCACAGATGTCCTCCACTCTCTGATTCCCCTGGTGCCCATGGACCTTGGAGGGACGCTGAGGATCAACCCGGAATCGCCTTGCACGACACGAAATCAATCTCCCTGCTGA >SEQIDNO: 50NLRGELDLLQRIQHSNIVSLVGFCIHEENRFIVYELMVNGSLETQLHGPSHGSALSWHIRMKIALDTARGLEYLHEHCNPPIIHRDLKSSNILLNSDFNAKISDFGLAVTSGNRSKGNLKLSGTLGYVAPEYLLDGKLTEKSDVYAFGVVLLELLLGRRPVEKMAPSQCQSIVTWAMPQLIDRSKLPTIIDPVIRDTMDRKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLIPLVPMDLGGTLRINPESPCTTRNQSPC >SEQIDNO: 51CGGGGGCTCTTATCACTCATTGCTGCTGCTACTGCACTGGGTACAAGCTTATTGCTCATGGGTTGCTTCTGGATTTATCATAGAAAGAAAATCCACAAATCTCATGACATTATTCATAGCCCAGATGTAGTTAAAGGTCTTGCATTATCCTCATATATTAGCAAATACAACTCCTTCAAGTCGAATTGTGTGAAACGACATGTCTCGTTGTGGGAGTACAATACACTCGAGTCGGCCACAAATAGTTTTCAAGAAAGCGAGATCTTGGGTGGAGGGGGGTTCGGGCTTGTGTACAAGGGAAAACTAGAAGACAACTTGTATGTAGCTGTGAAGAGGCTGGAAGTTGGAAGACAAAACGCAATTAAAGAATTCGAGGCTGAAATAGAGGTATTGGGCACGATTCAGCACCCGAATATAATTTCGTTGTTGGGATATAGCATTCATGCTGACACGAGGCTGCTAGTTTATGAACTGATGCAGAATGGATCTCTGGAGTATCAACTACATGGACCTTCCCATGGATCAGCATTAGCGTGGCATAATAGATTGAAAATCGCACTTGATACAGCAAGGGGATTAGAATATTTACATGAACATTGCAAACCACCAGTTATCCATAGAGATCTGAAATCCTCCAATATTCTTCTAGATGCCAACTTCAATGCCAAGATCTCAGATTTTGGTCTTGCTGTGCGCGATGGGGCTCAAAACAAAAATAACATTAAGCTCTCGGGAACCGTTGGCTATGTAGCTCCAGAATACCTATTAGATGGAATACTAACAGATAAAAGTGATGTTTATGGCTTCCGAGTTGTA >SEQIDNO: 52RGLLSLIAAATALGTSLLLMGCFWIYHRKKIHKSHDIIHSPDVVKGLALSSYISKYNSFKSNCVKRHVSLWEYNTLESATNSFQESEILGGGGFGLVYKGKLEDNLYVAVKRLEVGRQNAIKEFEAEIEVLGTIQHPNIISLLGYSIHADTRLLVYELMQNGSLEYQLHGPSHGSALAWHNRLKIALDTARGLEYLHEHCKPPVIHRDLKSSNILLDANFNAKISDFGLAVRDGAQNKNNIKLSGTVGYVAPEYLLDGILTDKSDVYGFRVV >SEQIDNO: 53GGGGATATACGTGTAGAATCAGCAACAAATAACTTCGGTGAAAGCGAGATATTAGGCGTAGGTGGATTTGGATGCGTGTATAAAGCTCGACTCGATGATAATTTGCATGTAGCTGTTAAAAGATTAGATGGTATTAGTCAAGACGCCATTAAAGAATTCCAGACGGAGGTGGATCTATTGAGTAAAATTCATCATCCGAATATCATCACCTTATTGGGATATTGTGTTAATGATGAAACCAAGCTTCTTGTTTATGAACTGATGCATAATGGATCTTTAGAAACTCAATTACATGGGCCTTCCAGTGGATCCAATTTAACATGGCATTGCAGGATGAAGATTGCTCTAGATACAGCAAGAGGATTAGAATATTTGCATGAGAACTGCAAACCATCGGTGATTCATAGAGATCTGAAATCATCTAATATCCTTCTGGATTCCAGCTTCAATGCTAAGCTTTCAGATTTTGGTCTTGCTATAATGGATGGGGCCCAGAACAAAAACAACATTAAGCTTTCAGGGACATTGGGTTATGTAGCTCCCGAGTATCTTTTAGATGGAAAATTGACGGATAAAAGTGACGTGTATGCGTTTGGAGTTGTGCTTTTAGAGCTTTTACTTGGAAGGCGACCTGTAGAAAAATTAGCAGAGTCGCAATGCCAATCTATTGTCACTTGGGCTATGCCACAATTAACAGACAGATCAAAGCTTCCGAATATTGTAGATCCCGTGATCAGATACACAATGGATCTCAAGCACCTGTACCAAGTTGCTGCGGTGGCTGTGTTATGTGTACAACCCGGACCAAGCTACCGGCCATTTATAAACCGACGTCTTGCATTCTCTGATCCCTCTTGTTCCCCGTGA >SEQIDNO: 54GDIRVESATNNFGESEILGVGGFGCVYKARLDDNLHVAVKRLDGISQDAIKEFQTEVDLLSKIHHPNIITLLGYCVNDETKLLVYELMHNGSLETQLHGPSSGSNLTWHCRMKIALDTARGLEYLHENCKPSVIHRDLKSSNILLDSSFNAKLSDFGLAIMDGAQNKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLAESQCQSIVTWAMPQLTDRSKLPNIVDPVIRYTMDLKHLYQVAAVAVLCVQPGPSYRPFINRRLAFSDPSCSP >SEQIDNO: 55AAGTTGAACTGTGAATGTCAATATGCTGAGAGAGAATTTGAGAATGAGGTGGATTTGTTAAGTAAAATTCAACATCCAAATGTAATTTCTCTACTGGGCTGTAGCAGTAATGAGGATTCAAGGTTTATTGTCTATGAGTTGATGCAAAATGGATCATTGGAAACTCAATTACATGGACCATCTCATGGCTCAGCATTGACTTGGCATATGAGGATGAAGATTGCTCTTGACACAGCTAGAGGTTTAAAATATCTGCATGAGCACTGCTACCCTGCAGTGATCCATAGAGATCTGAAATCTTCTAATATTCTTTTAGATGCAAACTTCAATGCCAAGCTTTCTGATTTTGGTCTTGCAATAACTGATGGGTCCCAAAACAAGAATAACATCAAGCTTTCAGGCACATTGGGGTATGTTGCCCCGGAGTATCTTTTAGATGGTAAATTGACAGATAAAAGTGATGTGTATGCTTTTGGAGTTGTGCTTCTTGAGCTTCTATTAGGAAGAAAGCCTGTGGAAAAACTTACACCATCTCAATGCCAGTCTATTGTCACATGGGCCATGCCACAGCTCACAGACAGATCCAAGCTTCCAAACATTGTGGATAATGTGATTAAGAATACAATGGATCCTAAGCACTTATACCAGGTTGCTGCTGTGGCTGTATTATGTGTGCAACCAGAGCCGTGCTACCGCCCTTTGATTGCAGATGTTCTACACTCCCTCATCCCTCTTGTACCTGTTGAGCTTGGAGGAACACTCAGAGTTGCACAAGTGACGCAGCAACCTAAGAATTCTAGTTAA >SEQIDNO: 56KLNCECQYAEREFENEVDLLSKIQHPNVISLLGCSSNEDSRFIVYELMQNGSLETQLHGPSHGSALTWHMRMKIALDTARGLKYLHEHCYPAVIHRDLKSSNILLDANFNAKLSDFGLAITDGSQNKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRKPVEKLTPSQCQSIVTWAMPQLTDRSKLPNIVDNVIKNTMDPKHLYQVAAVAVLCVQPEPCYRPLIADVLHSLIPLVPVELGGTLRVAQVTQQPKNSS >SEQIDNO: 57CAGTTGCATGGACCTCCTCGTGGATCAGCTTTGAATTGGCATCTTCGCATGGAAATTGCATTGGATGTGGCTAGGGGACTAGAATACCTCCATGAGCGCTGTAACCCCCCTGTAATCCATAGAGATCTCAAATCGTCTAATGTTCTATTGGATTCCTACTTCAATGCAAAGCTTTCTGACTTTTGGCCTAGCTATAGCTGGATGGAACTTAAACAAGAGCACCGTAAAGTCTTTCGGGAACTCTGGGATATGTGGCTCCAGAGTTACCTCTTAGATGGGAAATTAACTGATAAGAGTGATGTCTATGCTTTCGGCATTATACTTCTGGAGCTTCTAATGGGGAGAAGACCATTGGAGAAACTAGCAGGAGCTCAGTGCCAATCTATCGTCACATGGGCAATGCCACAGCTTACTGACAGGTCAAAGCTCCCAAATATTGTTGATCCTGTCATCAGAAACGGAATGGGCCTCAAGCACTTGTATCAAGTTGCTGCTGTAGCCGTGCTATGTGTACAACCAGAACCAAGTTACCGACCACTGATAACAGATGTCCTGCACTCCTTCATTCCCCTTGTACCAATTGAGCTTGGTGGGTCCTTGAGAGTTGTGGATTCTGCATTATCTGTTAACGCATAA >SEQIDNO: 58QLHGPPRGSALNWHLRMEIALDVARGLEYLHERCNPPVIHRDLKSSNVLLDSYFNAKLSDFWPSYSWMELKQEHRKVFRELWDMWLQSYLLDGKLTDKSDVYAFGIILLELLMGRRPLEKLAGAQCQSIVTWAMPQLTDRSKLPNIVDPVIRNGMGLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSFIPLVPIELGGSLRVVDSALSVNA >SEQIDNO: 59ATGGAGATGGCGCTAACTCCATTGCCGCTCCTGTGTTCGTCCGTCTTGTTCTTGGTGCTATCTTCGTGCTCGTTGGCCAATGGGAGGGATACGCCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTCCGGCGACGTCTACTGTGGCCACCGGCATTTCCGCCGCCGCCGCCGCCGCCGCCAATGGGACGGCCGCCTTGTCTTCGGCAGTTCCGGCGCCTCCGCCTGTTGTGATCGTAGTGCACCACCATTTCCACCGCGAGCTGGTCATCGCCGCCGTCCTCGCCTGCATCGCCACCGTCACGATCTTCCTTTCCACGCTCTACGCTTGGACACTATGGCGGCGATCTCGCCGGAGCACCGGCGGCAAGGTCACCAGGAGCTCAGACGCAGCGAAGGGGATCAAGCTGGTGCCGATCTTGAGCAGGTTCAACTCGGTGAAGATGAGCAGGAAGAGGCTGGTTGGGATGTTCGAGTACCCGTCGCTGGAGGCAGCGACAGAGAAGTTCAGCGAGAGCAACATGCTCGGTGTCGGCGGGTTTGGCCGCGTCTACAAGGCGGCGTTCGACGCCGGAGTTACCGCGGCGGTGAAGCGGCTCGACGGCGGCGGGCCCGACTGCGAGAAGGAATTCGAGAATGAGCTGGATTTGCTTGGCAGGATCAGGCACCCCAACATTGTGTCCCTCTTGGGCTTCTGTATCCATGAGGGGAATCACTACATTGTTTATGAGCTGATGGAGAAGGGATCACTGGAAACACAGCTTCATGGGTCTTCACATGGATCAACTCTGAGCTGGCACATCCGGATGAAGATCGCCCTTGACACGGCCAGGGGATTAGAGTACCTTCATGAGCACTGCAGTCCACCAGTGATCCATAGGGATCTGAAATCGTCTAACATACTTTTGGATTCAGACTTCAATGCTAAGATTGCAGATTTTGGTCTTGCTGTGTCTAGTGGGAGTGTCAACAAAGGGAGTGTGAAGCTCTCCGGGACCTTGGGTTATGTAGCTCCTGAGTACTTGTTGGATGGGAAGTTGACTGAAAAGAGCGATGTATACGCGTTCGGAGTAGTGCTTCTAGAGCTCCTTATGGGGAGGAAGCCTGTTGAGAAGATGTCACCATCTCAGTGCCAATCAATTGTGACATGGGCAATGCCACAGTTGACCGACAGATCGAAGCTCCCCAGCATAGTTGACCCAGTGATCAAGGACACCATGGATCCAAAACACCTGTACCAAGTTGCAGCAGTGGCTGTTCTATGCGTGCAGGCTGAACCAAGCTACAGGCCACTGATCACAGATGTGCTCCACTCTCTTGTTCCTCTAGTGCCGACGGAGCTCGGAGGAACACTAAGAGCTGGAGAGCCACCTTCCCCGAACCTGAGGAATTCTCCATGCTGA >SEQIDNO: 60MEMALTPLPLLCSSVLFLVLSSCSLANGRDTPSSSSSSSSSSSSSSSSSSSSSSPATSTVATGISAAAAAAANGTAALSSAVPAPPPVVIVVHHHFHRELVIAAVLACIATVTIFLSTLYAWTLWRRSRRSTGGKVTRSSDAAKGIKLVPILSRFNSVKMSRKRLVGMFEYPSLEAATEKFSESNMLGVGGFGRVYKAAFDAGVTAAVKRLDGGGPDCEKEFENELDLLGRIRHPNIVSLLGFCIHEGNHYIVYELMEKGSLETQLHGSSHGSTLSWHIRMKIALDTARGLEYLHEHCSPPVIHRDLKSSNILLDSDFNAKIADFGLAVSSGSVNKGSVKLSGTLGYVAPEYLLDGKLIEKSDVYAFGVVLLELLMGRKPVEKMSPSQCQSIVTWAMPQLTDRSKLPSIVDPVIKDTMDPKHLYQVAAVAVLCVQAEPSYRPLITDVLHSLVPLVPTELGGTLRAGEPPSPNLRNSPC >SEQIDNO: 61TACTCTCTTTTACAAACTGCTACGAACAACTTCAGCTCCTCCAATTTGCTGGGCGAGGGAAGTTTCGGGCATGTGTATAAAGCGAGACTCGATTATGATGTCTATGCCGCTGTAAAGAGACTTACCAGCGTAGGAAAACAGCCCCAAAAAGAACTCCAGGGAGAGGTGGATCTGATGTGCAAGATAAGACATCCCAACTTGGTGGCTCTCCTGGGCTATTCAAATGACGGCCCAGAGCCCTTGGTTGTGTACGAGCTCATGCAGAATGGTTCACTTCATGATCAGCTTCATGGCCCCTCATGCGGGAGTGCACTCACCTGGTACCTACGACTAAAGATTGCTCTTGAAGCTGCCAGCAGAGGACTGGAGCACCTGCATGAAAGCTGCAAGCCTGCAATAATCCACAGAGACTTCAAGGCATCCAACATCCTCTTGGACGCCAGCTTCAATGCGAAGGTGTCCGACTTTGGTATAGCGGTAGCTCTGGAGGAAGGTGGCGTGGTGAAAGACGACGTACAAGTGCAAGGCACCTTCGGGTACATTGCTCCTGAGTACCTGATGGACGGGACATTGACAGAGAAGAGTGATGTTTACGGATTTGGAGTAGTATTGCTTGAGCTGCTGACAGGCAGACTGCCCATTGATACGTCCTTACCACTCGGATCGCAATCTCTAGTGACATGGGTAACACCCATACTAACTAACCGAGCAAAGCTGATGGAAGTTATCGACCCCACCCTTCAAGATACGCTGAACGTGAAGCAACTTCACCAGGTGGCCGCAGTGGCAGTCCTTTGCGTCCAAGCGGAACCCAGCTACCGCCCTCTCATCGCCGACGTGGTTCAGTCACTGGCTCCGCTGGTGCCTCAAGAGCTCGGCGGC GCATTGCGA >SEQIDNO: 62YSLLQTATNNFSSSNLLGEGSFGHVYKARLDYDVYAAVKRLTSVGKQPQKELQGEVDLMCKIRHPNLVALLGYSNDGPEPLVVYELMQNGSLHDQLHGPSCGSALTWYLRLKIALEAASRGLEHLHESCKPAIIHRDFKASNILLDASFNAKVSDFGIAVALEEGGVVKDDVQVQGTFGYIAPEYLMDGTLIEKSDVYGFGVVLLELLTGRLPIDTSLPLGSQSLVTWVTPILTNRAKLMEVIDPTLQDTLNVKQLHQVAAVAVLCVQAEPSYRPLIADVVQSLAPLVPQELGG ALR >SEQIDNO: 63ACCTCAGATGCCTATAGGGGTATTCCACTCATGCCTCTCCTGAATCGTTTGAACTCCCGTATTTCCAAGAAGAAGGGATGTGCAACTGCAATTGAATATTCTAAGCTGCAAGCAGCTACAAATAACTTCAGCAGCAATAACATTCTTGGAGAGGGTGGATTTGCGTGTGTATACAAGGCCATGTTTGATGATGATTCCTTTGCTGCTGTGAAGAAGCTAGATGAGGGTAGCAGACAGGCTGAGCATGAATTTCAGAATGAAGTGGAGCTGATGAGCAAAATCCGACATCCAAACCTTGTTTCTTTGCTTGGGTTCTGCTCTCATGAAAATACACGGTTCTTAGTATATGATCTGATGCAGAATGGCTCTTTGGAAGACCAATTACATGGGCCATCTCACGGATCTGCACTTACATGGTTTTTGCGCATAAAGATAGCACTTGATTCAGCAAGGGGTCTAGAACACTTGCATGAGCACTGCAACCCTGCAGTGATTCATCGAGATTTCAAATCATCAAATATTCTTCTTGATGCAAGCTTCAACGCCAAGCTTTCAGATTTTGGTCTTGCAGTAACAAGTGCAGGATGTGCTGGCAATACAAATATTGATCTAGTAGGGACATTGGGATATGTAGCTCCAGAATACCTACTTGATGGTAAATTGACAGAGAAAAGTGATGTCTATGCATATGGAGTTGTTTTGTTGGAGCTACTTTTTGGAAGAAAGCCAATTGATAAATCTCTACCAAGTGAATGCCAATCTCTCATTTCTTGGGCAATGCCACAGCTAACAGATAGAGAAAAGCTCCCAACTATAGTAGACCCCATGATCAAAGGCACAATGAACTTGAAACACCTATATCAAGTAGCAGCTGTTGCAATGCTATGTGTGCAGCCAGAACCCAGTTACAGGCCATTAATAGCTGACGTTGTGCACTCTCTCATTCCTCTCGTACCAATAGAACTCGGGGGAACTTTAAAGCTCTCTAATGCACGACCCACTGAGATGAAGTTATTTACTTCTTCCCAATGCAGTGTTGAGATTGCTTCCAACCCAAAATTGTGA >SEQIDNO: 64TSDAYRGIPLMPLLNRLNSRISKKKGCATAIEYSKLQAATNNFSSNNILGEGGFACVYKAMFDDDSFAAVKKLDEGSRQAEHEFQNEVELMSKIRHPNLVSLLGFCSHENTRFLVYDLMQNGSLEDQLHGPSHGSALTWFLRIKIALDSARGLEHLHEHCNPAVIHRDFKSSNILLDASFNAKLSDFGLAVTSAGCAGNTNIDLVGTLGYVAPEYLLDGKLTEKSDVYAYGVVLLELLFGRKPIDKSLPSECQSLISWAMPQLTDREKLPTIVDPMIKGTMNLKHLYQVAAVAMLCVQPEPSYRPLIADVVHSLIPLVPIELGGTLKLSNARPTEMKLFTSSQCSVEIASNPKL >SEQIDNO: 65AATTCGGCACGAGGAGAACACTTGCACGAGCACTGCAACCCTGCAGTGATTCACCGAGATTTCAAATCATCAAATATTCTTCTTGATGCAAGCTTCAACGCCAAGCTTTCAGATTTTGGTCTTGCAGTAAAAAGTGCAGGATGTGCTGGTAACACAAATATTGATCTAGTAGGGACATTGGGATATGTAGCTCCAGAATACATGCTTGATGGTAAATTGACAGAGAAAAGTGATGTCTATGCATATGGAGTTGTTTTGTTAGAGCTACTTTTTGGAAGAAAGCCAATTGATAAATCTCTACCAAGTGAATGCCAATCTCTCATTTCTTGGGCAATGCCACAGCTAACAGATAGAGAAAAGCTCCCGACTATAATAGATCCCATGATCAAAGGCGCAATGAACTTGAAACACCTATATCAAGTGGCAGCTGTTGCAGTGCTATGTGTGCAGCCAGAACCCAGTTACAGGCCATTAATAGCTGACGTTGTGCACTCTCTCATTCCTCTCGTACCAGTAGAACTTGGGGGAACATTAAAGTCATCACCCACTGAGATGAAGTCATTTGCTTCTTCCCAATGCAGTGCCCAC GTTGCTTC >SEQIDNO: 66NSARGEHLHEHCNPAVIHRDFKSSNILLDASFNAKLSDFGLAVKSAGCAGNTNIDLVGTLGYVAPEYMLDGKLIEKSDVYAYGVVLLELLFGRKPIDKSLPSECQSLISWAMPQLTDREKLPTIIDPMIKGAMNLKHLYQVAAVAVLCVQPEPSYRPLIADVVHSLIPLVPVELGGTLKSSPTEMKSFASSQCSAH VAS >SEQIDNO: 67ATGTTCTTGTTTCCTAAAACAGTTCCTATTTGGTTTTTTCATCTGTGTCTAGTAGCAGTTCATGCCATACAAGAAGACCCACCTGTCCCTTCACCATCTCCCTCTCTCATTTCTCCTATTTCAACTTCAATGGCTGCCTTCTCTCCAGGGGTTGAATCGGAAATGGGAATCAAAGACCACCCCCAGCATGATGACCTCCACAGGAAAATAATCTTGTTGCTCACTGTTGCTTGTTGCATACTTGTTATCATCCTTCTTTCTTTGTGTTCTTGTTTCATTTACTATAAGAAGTCCTCACAAAAGAAAAAAGCTACTCGGTGTTCAGATGTGGAGAAAGGGCTTTCATTGGCACCATTTTTGGGCAAATTCAGTTCCTTGAAAATGGTTAGTAATAGGGGATCTGTTTCATTAATTGAGTATAAGATACTAGAGAAAGGAACAAACAATTTTGGCGATGATAAATTGTTGGGAAAGGGAGGATTTGGACGTGTATATAAGGCTGTAATGGAAGATGACTCAAGTGCTGCAGTCAAGAAACTAGACTGCGCAACTGATGATGCGCAGAGAGAATTTGAGAATGAGGTGGATTTGTTAAGCAAATTTCACCATCCAAATATAATTTCTATTGTGGGTTTTAGTGTTCATGAGGAGATGGGGTTCATTATTTATGAGTTAATGCCAAATGGGTGCCTTGAAGATCTACTGCATGGACCTTCTCGTGGATCTTCACTAAATTGGCATTTAAGGTTGAAAATTGCTCTTGATACAGCAAGAGGATTAGAATATCTGCATGAATTCTGCAAGCCAGCAGTGATCCATAGAGATCTGAAATCATCGAATATTCTTTTGGACGCCAACTTCAATGCCAAGCTGTCAGATTTTGGTCTTGCTGTAGCTGATAGCTCTCATAACAAGAAAAAGCTCAAGCTTTCAGGCACTGTGGGTTATGTAGCCCCAGAGTATATGTTAGATGGTGAATTGACGGATAAGAGTGATGTCTATGCTTTTGGAGTTGTGCTTCTAGAGCTTCTATTAGGAAGAAGGCCTGTAGAAAAACTGACACCAGCTCATTGCCAATCTATAGTAACATGGGCCATGCCTCAGCTCACTAACAGAGCTGTGCTTCCAACCCTTGTGGATCCTGTGATCAGAGATTCAGTAGATGAGAAGTACTTGTTCCAGGTTGCAGCAGTAGCCGTGTTGTGTATTCAACCAGAGCCAAGTTACCGCCCTCTCATAACAGATGTTGTGCACTCTCTCGTCCCATTAGTTCCTCTTGAGCTTGGAGGGACACTAAGAGTTCCACAGCCTACAACTCCCAGAGGTCAACGACAAGGCCCATCAAAGAAACTGTTTTTGGATGGTGCTGCCT CTGCT >SEQIDNO: 68MFLFPKTVPIWFFHLCLVAVHAIQEDPPVPSPSPSLISPISTSMAAFSPGVESEMGIKDHPQHDDLHRKIILLLTVACCILVIILLSLCSCFIYYKKSSQKKKATRCSDVEKGLSLAPFLGKFSSLKMVSNRGSVSLIEYKILEKGTNNFGDDKLLGKGGFGRVYKAVMEDDSSAAVKKLDCATDDAQREFENEVDLLSKFHHPNIISIVGFSVHEEMGFIIYELMPNGCLEDLLHGPSRGSSLNWHLRLKIALDTARGLEYLHEFCKPAVIHRDLKSSNILLDANFNAKLSDFGLAVADSSHNKKKLKLSGTVGYVAPEYMLDGELTDKSDVYAFGVVLLELLLGRRPVEKLTPAHCQSIVTWAMPQLTNRAVLPTLVDPVIRDSVDEKYLFQVAAVAVLCIQPEPSYRPLITDVVHSLVPLVPLELGGTLRVPQPTTPRGQRQGPSKKLFLDGAASA >SEQIDNO: 69GCTGCTGCGGTGAAGAGATTGGATGGTGGGGCTGGGGCACATGATTGCGAGAAGGAATTCGAGAATGAGTTAGATTTGCTTGGAAAGATTCGGCATCCGAACATTGTGTCCCTTGTGGGCTTCTGTATTCATGAGGAGAACCGTTTCATTGTTTATGAGCTGATAGAGAATGGGTCGTTGGATTCACAACTTCATGGGCCATCACATGGTTCAGCTCTGAGCTGGCATATTCGGATGAAGATTGCTCTTGACACGGCAAGGGGATTAGAGTACCTGCATGAGCACTGCAACCCACCAGTTATCCATAGGGATCTGAAGTCATCTAACATACTTTTAGATTCAGACTTCAGTGCTAAGATTTCAGATTTTGGCCTTGCGGTGATTAGTGGGAATCACAGCAAAGGGAATTTAAAGCTTTCTGGGACTATGGGCTATGTGGCCCCTGAGTACTTATTGGATGGGAAGTTGACTGAGAAGAGCGATGTATATGCGTTTGGGGTGGTACTTCTAGAACTTCTACTGGGAAGGAAACCTGTTGAGAAGATGGCACAATCTCAATGCCAATCAATTGTTACATGGGCCATGCCTCAGCTAACTGATAGATCCAAACTCCCTAACATAATTGATCCCATGATCAAGAACACAATGGATCTGAAACACTTGTACCAAGTTGCTGCAATGGCTGT GCTCTGA >SEQIDNO: 70AAAVKRLDGGAGAHDCEKEFENELDLLGKIRHPNIVSLVGFCIHEENRFIVYELIENGSLDSQLHGPSHGSALSWHIRMKIALDTARGLEYLHEHCNPPVIHRDLKSSNILLDSDFSAKISDFGLAVISGNHSKGNLKLSGTMGYVAPEYLLDGKLTEKSDVYAFGVVLLELLLGRKPVEKMAQSQCQSIVTWAMPQLTDRSKLPNIIDPMIKNTMDLKHLYQVAAMAVL >SEQIDNO: 71ACCCTCGGTTATGTAGCTCCTGAGTATCTGTTAGATGGTAAGTTAACAGAGAAAAGCGATGTGTATGGGTTTGGAGTAGTGTTACTCGAGCTTCTGCTTGGGAAGAAGCCTATGGAGAAAGTGGCAACAACAGCAACTCAGTGCCAGATGATAGTCACATGGACCATGCCTCAGCTCACTGACAGAACGAAACTTCCGAATATCGTGGATCCGGTGATCAGAAACTCCATGGATTTAAAGCACTTGTACCAGGTTGCTGCTGTGGCAGTATTGTGTGTGCAGCCAGAACCGAGTTATCGGCCATTGATAACTGATATTTTGCATTCTCTTGTGCCCCTTGTCCCTGTTGAGCTTGGTGGGACGCTCAGGAACTCGATAACAATGGCTACAACAACAATATCTCCTGAAAGCTAA >SEQIDNO: 72TLGYVAPEYLLDGKLILKSDVYGFGVVLLELLLGKKPMEKVATTATQCQMIVTWTMPQLTDRTKLPNIVDPVIRNSMDLKHLYQVAAVAVLCVQPEPSYRPLITDILHSLVPLVPVELGGTLRNSITMATTTISPES >SEQIDNO: 73CGGCACGAGGGGCTGGTGGCCATGATCGAGTACCCGTCGCTGGAGGCGGCGACGGGCAAGTTCAGCGAGAGCAACGTGCTCGGCGTCGGCGGGTTCGGCTGCGTCTACAAGGCGGCGTTCGACGGCGGCGCCACCGCCGCCGTGAAGAGGCTCGAAGGCGGCGAGCCGGACTGCGAGAAGGAGTTCGAGAATGAGCTGGACTTGCTTGGCAGGATCAGGCACCCAAACATAGTGTCCCTCCTGGGCTTCTGCGTCCATGGTGGCAATCACTACATTGTTTATGAGCTCATGGAGAAGGGATCATTGGAGACACAACTGCATGGGCCTTCACATGGATCGGCTATGAGCTGGCACGTCCGGATGAAGATCGCGCTCGACACGGCGAGGGGATTAGAGTATCTTCATGAGCACTGCAATCCACCAGTCATCCATAGGGATCTGAAATCGTCTAATATACTCTTGGATTCAGACTTCAATGCTAAGATTGCAGATTTTGGCCTTGCAGTGACAAGTGGGAATCTTGACAAAGGGAACCTGAAGATCTCTGGGACCTTGGGATATGTAGCTCCCGAGTACTTATTAGATGGGAAGTTGACCGAGAAGAGCGACGTCTACGCGTTTGGAGTAGTGCTTCTAGAGCTCCTGATGGGGAGGAAGCCTGTTGAGAAGATGTCACCATCTCAGTGCCAATCAATTGTGTCATGGGCCATGCCTCAGCTAACCGACAGATCGAAGCTACCCAACATCATCGACCCGGTGATCAAGGACACAATGGACCCAAAGCATTTATACCAAGTTGCGGCGGTGGCCGTTCTATGCGTGCAGCCCGAACCGAGTTACAGACCGCTGATAACAGACGTTCTCCACTCCCTTGTTCCTCTGGTACCCGCGGATCTCGGGGGGAACGCTCAGAGTTACAGAGCCGCATTCTCCACACCAAATGTACCATCCCTCTTGAGAAGTGATCCTACAAGTTTCGTCGAAGCGGGGAAAGCGAATNTATACGGTCCAGCGGTAGATGGCTGTTATTTTGGTACTTATATCTCACCCTGTCCTGCTGCTTATCTTAGGATGAGTGANGAGCTCCNACCTGCTGCTTTTGCTGGTTGGGCAGAGAGAATACAGTTCTGGTTAGGATTG >SEQIDNO: 74RHEGLVAMIEYPSLEAATGKFSESNVLGVGGFGCVYKAAFDGGATAAVKRLEGGEPDCEKEFENELDLLGRIRHPNIVSLLGFCVHGGNHYIVYELMEKGSLETQLHGPSHGSAMSWHVRMKIALDTARGLEYLHEHCNPPVIHRDLKSSNILLDSDFNAKIADFGLAVTSGNLDKGNLKISGTLGYVAPEYLLDGKLTEKSDVYAFGVVLLELLMGRKPVEKMSPSQCQSIVSWAMPQLTDRSKLPNIIDPVIKDTMDPKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPADLGGNAQSYRAAFSTPNVPSLLRSDPTSFVEAGKANXYGPAVDGCYFGTYISPCPAAYLRMSXELXPAAFAGWAERIQFWLGL >SEQIDNO: 75ATGAAAGTGATTGGGAGAAAGGGTTATGTCTCTTTTATTGATTATAAGGTACTAGAAACTGCAACAAACAATTTTCAGGAAAGTAATATCCTGGGTGAGGGCGGGTTTGGTTGCGTCTACAAGGCGCGGTTGGATGATAACTCCCATGTGGCTGTGAAGAAGATAGATGGTAGAGGCCAGGATGCTGAGAGAGAATTTGAGAATGAGGTGGATTTGTTGACTAAAATTCAGCACCCAAATATAATTTCTCTCCTGGGTTACAGCAGTCATGAGGAGTCAAAGTTTCTTGTCTATGAGCTGATGCAGAATGGATCTCTGGAAACTGAATTGCACGGACCTTCTCATGGATCATCTCTAACTTGGCATATTCGAATGAAAATCGCTCTGGATGCAGCAAGAGGATTAGAGTATCTACATGAGCACTGCAACCCACCAGTCATCCATAGAGATCTTAAATCATCTAATATTCTTCTGGATTCAAACTTCAATGCCAAGCTTTCGGATTTTGGTCTAGCTGTAATTGATGGGCCTCAAAACAAGAACAACTTGAAGCTTTCAGGCACCCTGGGTTATCTAGCTCCTGAGTATCTTTTAGATGGTAAACTGACTGATAAGAGTGATGTGTATGCATTTGGAGTGGTGCTTCTAGAGCTACTACTGGGAAGAAAGCCTGTGGAAAAACTGGCACCAGCTCAATGCCAGTCCATTGTCACATGGGCCATGCCACAGCTGACTGACAGATCAAAGCTCCCAGGCATCGTTGACCCTGTGGTCAGAGACACGATGGATCTAAAGCATTTATACCAAGTTGCTGCTGTAGCTGTGCTATGTGTGCAACCAGAACCAAGTTACCGGCCATTGATAACAGATGTTCTGCACTCCCTCATCCCACTCGTTCCAGTTGAGTTGGGAGGGATGCTAAAAGTTACCCAGCAAGCGCCGCCTATCAACACCACTGCACCTTCTGCTGGAGGTTGA >SEQIDNO: 76MKVIGRKGYVSFIDYKVLETATNNFQESNILGEGGFGCVYKARLDDNSHVAVKKIDGRGQDAEREFENEVDLLTKIQHPNIISLLGYSSHEESKFLVYELMQNGSLETELHGPSHGSSLTWHIRMKIALDAARGLEYLHEHCNPPVIHRDLKSSNILLDSNFNAKLSDFGLAVIDGPQNKNNLKLSGTLGYLAPEYLLDGKLTDKSDVYAFGVVLLELLLGRKPVEKLAPAQCQSIVTWAMPQLTDRSKLPGIVDPVVRDTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLIPLVPVELGGMLKVTQQAPPINTTAPSAGG >SEQIDNO: 77ATGCCGCCGCCATCGCCGCTCCTCCGTTCCTCCGCCTTCGTCGTCTTGCTGCTCCTGGTGTGTCGCCCGTTGTTGGTCGCCAATGGGAGGGCCACGCCGCCTTCTCCGGGATGGCCACCGGCGGCTCAGCCCGCGCTGCAGCCTGCACCCACCGCCAGCGGCGGCGTGGCCTCCGTGCTTCCTTCGGCCGTGGCGCCTCCTCCCTTAGGTGTGGTTGTGGCGGAGAGGCACCACCACCTCAGCAGGGAGCTCGTCGCTGCCATTATCCTCTCATCCGTCGCCAGCGTCGTGATCCCCATTGCCGCGCTGTATGCCTTCTTGCTGTGGCGACGATCACGGCGAGCCCTGGTGGATTCCAAGGACACCCAGAGCATAGATACCGCAAGGATTGCTTTTGCGCCGATGTTGAACAGCTTTGGCTCGTACAAGACTACCAAGAAGAGTGCCGCGGCGATGATGGATTACACATCTTTGGAGGCAGCGACAGAAAACTTCAGTGAGAGCAATGTCCTTGGATTTGGTGGGTTTGGGTCTGTGTACAAAGCCAATTTTGATGGGAGGTTTGCTGCTGCGGTGAAGAGACTGGATGGTGGGGCACATGATTGCAAGAAGGAATTCGAGAATGAGCTAGACTTGCTTGGGAAGATTCGACATCCGAACATCGTGTCCCTTGTGGGCTTCTGCATTCATGAGGAGAACCGTTTCGTTGTTTATGAGCTGATGGAGAGTGGGTCGTTGGATTCGCAACTTCATGGGCCATCACATGGTTCAGCTCTGAGCTGGCATATTCGGATGAAGATTGCTCTCGACACAGCAAGGGGATTAGAGTACCTGCATGAGCACTGCAACCCACCGGTTATCCATAGGGATCTTAAGTCATCTAACATACTTTTAGATTCAGACTTCAGCGCTAAGATTTCAGACTTTGGCCTGGCAGTGACTAGTGGGAATCACAGCAAAGGGAATTTAAAGCTTTCTGGGACTATGGGCTATGTGGCTCCTGAGTACTTATTAGATGGGAAGCTGACTGAGAAGAGCGATGTATACGCGTTTGGGGTAGTACTTCTAGAACTCCTGCTGGGAAGGAAACCTGTCGAGAAGATGGCACAATCTCAGTGCCGATCAATCGTTACATGGGCCATGCCTCAGCTAACTGATAGATCCAAGCTCCCGAACATAATTGATCCCATGATCAAGAACACAATGGATCTGAAACACTTGTACCAAGTTGCTGCAGTGGCCGTGCTCTGCGTGCAGCCAGAGCCGAGTTACAGGCCACTGATCACCGACGTGCTTCACTCACTGGTACCTCTAGTGCCCACGGAGCTTGGAGGAACGCTGAGGATCGGCCCGGAATCGCCCTACCTACGCT ACTAA >SEQIDNO: 78MPPPSPLLRSSAFVVLLLLVCRPLLVANGRATPPSPGWPPAAQPALQPAPTASGGVASVLPSAVAPPPLGVVVAERHHHLSRELVAAIILSSVASVVIPIAALYAFLLWRRSRRALVDSKDTQSIDTARIAFAPMLNSFGSYKTTKKSAAAMMDYTSLEAA1ENFSESNVLGFGGFGSVYKANFDGRFAAAVKRLDGGAHDCKKEFENELDLLGKIRHPNIVSLVGFCIHEENRFVVYELMESGSLDSQLHGPSHGSALSWHIRMKIALDTARGLEYLHEHCNPPVIHRDLKSSNILLDSDFSAKISDFGLAVTSGNHSKGNLKLSGTMGYVAPEYLLDGKLTEKSDVYAFGVVLLELLLGRKPVEKMAQSQCRSIVTWAMPQLTDRSKLPNIIDPMIKNTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPIELGGTLRIGPESPYLRY >SEQIDNO: 79ATGTTGCTCGCGTGTCCTGCAGTGATCATCGTGGAGCGCCACCGTCATTTCCACCGTGAGCTAGTCATCGCCTCCATCCTCGCCTCAATCGCCATGGTCGCGATTATCCTCTCCACGCTGTACGCGTGGATCCCGCGCAGGCGGTCCCGCCGGCTGCCCCGCGGCATGAGCGCAGACACCGCGAGGGGGATCATGCTGGCGCCGATCCTGAGCAAGTTCAACTCGCTCAAGACGAGCAGGAAGGGGCTCGTGGCGATGATCGAGTACCCGTCGCTGGAGGCAGCGACAGGGGGGTTCAGTGAGAGCAACGTGCTCGGCGTAGGCGGCTTCGGTTGCGTCTACAAGGCAGTCTTCGATGGCGGCGTTACCGCGGCGGTCAAGAGGCTGGAGGGAGGTGGCCCTGAGTGCGAGAAGGAATTCGAGAATGAGCTGGATCTGCTTGGCAGGATTCGGCACCCCAACATCGTGTCCCTGCTGGGCTTTTGTGTTCACGAGGGGAATCACTACATTGTTTATGAGCTCATGGAGAAGGGATCCCTGGACACACAGCTGCATGGGGCCTCACATGGATCAGCGCTGACCTGGCATATCCGGATGAAGATCGCACTCGACATGGCCAGGGGATTAGAATACCTCCATGAGCACTGCAGTCCACCAGTGATCCATAGGGATCTGAAGTCATCTAACATACTTTTAGATTCTGACTTCAATGCTAAGATTTCAGATTTTGGTCTTGCAGTGACCAGTGGGAACATTGACAAGGGAAGCATGAAGCTTTCTGGGACCTTGGGTTATGTGGCCCCTGAGTACCTATTAGATGGGAAGCTGACTGAAAAGAGTGACGTATATGCATTTGGAGTGGTGCTTCTTGAGCTACTAATGGGAAGGAAGCCTGTCGAGAAGATGAGTCAAACTCAGTGCCAATCAATTGTGACGTGGGCCATGCCGCAGCTGACTGACAGAACAAAACTTCCCAACATAGTTGACCCAGTGATCAGGGACACCATGGATCCAAAGCATTTGTACCAAGTGGCAGCAGTGGCAGTTCTATGTGTGCAACCAGAACCAAGTTACAGACCGCTGATTACTGATGTTCTCCACTCTCTTGTCCCTCTAGTCCCTGTGGAGCTCGGAGGGACACTGAGGGTTGTAGAGCCACCTTCCCCAAACCTAAAACATTCTCCTTGT >SEQIDNO: 80MLLACPAVIIVERHRHFHRELVIASILASIAMVAIILSTLYAWIPRRRSRRLPRGMSADTARGIMLAPILSKFNSLKTSRKGLVAMIEYPSLEAATGGFSESNVLGVGGFGCVYKAVFDGGVTAAVKRLEGGGPECEKEFENELDLLGRIRHPNIVSLLGFCVHEGNHYIVYELMEKGSLDTQLHGASHGSALTWHIRMKIALDMARGLEYLHEHCSPPVIHRDLKSSNILLDSDFNAKISDFGLAVTSGNIDKGSMKLSGTLGYVAPEYLLDGKLTEKSDVYAFGVVLLELLMGRKPVEKMSQTQCQSIVTWAMPQLTDRTKLPNIVDPVIRDTMDPKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRVVEPPSPNLK HSPC >SEQIDNO: 81ATGAAGAAGAAGCTTGTGCTGCATCTGCTTCTTTTCCTTGTTTGTGCTCTTGAAAACATTGTTTTGGCCGTACAAGGCCCTGCTTCATCACCCATTTCTACTCCCATCTCTGCTTCAATGGCTGCCTTCTCTCCAGCTGGGATTCAACTTGGAGGTGAGGAGCACAAGAAAATGGATCCAACCAAGAAAATGTTATTAGCTCTCATTCTTGCTTGCTCTTCATTGGGTGCAATTATCTCTTCCTTGTTCTGTTTATGGATTTATTACAGGAAGAATTCAAGCAAATCCTCTAAAAATGGCGCTAAGAGCTCAGATGGTGAAAAAGGGAATGGTTTGGCACCATATTTGGGTAAATTCAAGTCTATGAGGACGGTTTCCAAAGAGGGTTATGCTTCGTTTATGGACTATAAGATACTTGAAAAAGCTACAAACAAGTTCCATCATGGTAACATTCTGGGTGAGGGTGGATTTGGATGTGTTTACAAGGCTCAATTCAATGATGGTTCTTATGCTGCTGTTAAGAAGTTGGACTGTGCAAGCCAAGATGCTGAAAAAGAATATGAGAATGAGGTGGGTTTGCTATGTAGATTTAAGCATTCCAATATAATTTCACTGTTGGGTTATAGCAGTGATAACGATACAAGGTTTATTGTTTATGAGTTGATGGAAAATGGTTCTTTGGAAACTCAATTACATGGACCTTCTCATGGTTCATCATTAACTTGGCATAGGAGGATGAAAATTGCTTTGGATACAGCAAGAGGATTAGAATATCTACATGAGCATTGCAATCCACCAGTCATCCATAGAGATCTGAAATCATCTAATATACTTTTGGATTTGGACTTCAATGCAAAGCTTTCAGATTTTGGTCTTGCAGTAACTGATGCGGCAACAAACAAGAATAACTTGAAGCTTTCGGGTACTTTAGGTTATCTAGCTCCAGAATACCTTTTAGATGGTAAATTAACAGATAAGAGTGATGTTTATGCATTCGGTGTTGTGCTGCTCGAACTTCTATTGGGACGAAAGGCTGTTGAAAAATTATCACAACTCAGTGCCAATCTTAGGTCCATTT GGGCATAG >SEQIDNO: 82MKKKLVLHLLLFLVCALENIVLAVQGPASSPISTPISASMAAFSPAGIQLGGEEHKKMDPTKKMLLALILACSSLGAIISSLFCLWIYYRKNSSKSSKNGAKSSDGEKGNGLAPYLGKFKSMRTVSKEGYASFMDYKILEKATNKFHHGNILGEGGFGCVYKAQFNDGSYAAVKKLDCASQDAEKEYENEVGLLCRFKHSNIISLLGYSSDNDTRFIVYELMENGSLETQLHGPSHGSSLTWHRRMKIALDTARGLEYLHEHCNPPVIHRDLKSSNILLDLDFNAKLSDFGLAVTDAATNKNNLKLSGTLGYLAPEYLLDGKLTDKSDVYAFGVVLLELLLGRKAVEKLSQLSANLRSIWA >SEQIDNO: 83GGAGTGGGAATTGAGAAGCAGCCACCCACCCACCCACCCTATGGATAAAAATAGAAGGCTGTTGATAGCACTCATTGTAGCTTCTACTGCATTAGGACTAATCTTTATCTTCATCATTTTATTCTGGATTTTTCACAAAAGATTTCACACCTCAGATGTTGTGAAGGGAATGAGTAGGAAAACATTGGTTTCTTTAATGGACTACAACATACTTGAATCAGCCACCAACAAATTTAAAGAAACTGAGATTTTAGGTGAGGGGGGTTTTGGATGTGTGTACAAAGCTAAATTGGAAGACAATTTTTATGTAGCTGTCAAGAAACTAACCCAAAATTCCATTAAAGAATTTGAGACTGAGTTAGAGTTGTTGAGTCAAATGCAACATCCCAATATTATTTCATTGTTGGGATATTGCATCCACAGTGAAACAAGATTGCTTGTCTATGAACTCATGCAAAATGGATCACTAGAAACTCAATTACATGGGCCTTCCCGTGGATCAGCATTAACTTGGCATCGCAGGATAAAAATTGCCCTTGATGCAGCAAGAGGAATAGAATATTTACATGAGCAGCGCCATCCCCCTGTAATTCATAGAGATCTGAAATCATCTAATATTCTTTTAGATTCCAACTTCAATGCAAAGGTAAAACTTTTTATGTAGAAATTATACTAGGACTAGTTTTCCCTCTATTAATCTTGTGTTGTGATTAATTTTAGCTGTCAGATTTTGGTCTTGCTGTGTTGAGTGGGGCTCAAAACAAAAACAATATCAAGCTTTCTGGAACTATAGGTTATGTAGCGCCTGAATACATGTTAGATGGAAAATTAAGTGATAAAAGTGATGTTTATGGTTTTGGAGTAGTACTTTTGGAGCTGTTATTGGGAAGGCGGCCTGTAGAAAAGGAGGCAGCCACTGAATGTCAGTCTATAGTGACATGGGCCATGCCTCAGCTGACAGATAGATCAAAGCTTCCAAACATTGTTGATCCTGTCATACAAAACACAATGGATTTAAAGCATNTGTATCAGGTTGCTGCAGGTGCTCTATTATGTGTTCAGCCAGAGCCAAGCTATCGTCCCGTATAA >SEQIDNO: 84GAGTATCAGTTATTGGAAGCTGCAACTGACAATTTTAGTGAGAGTAATATTTTGGGAGAAGGTGGATTTGGATGTGTTTACAAAGCATGTTTTGATAACAACTTTCTCGCTGCTGTCAAGAGAATGGATGTTGGTGGGCAAGATGCAGAAAGAGAATTTGAGAAAGAAGTAGATTTGTTGAATAGAATTCAGCATCCGGATATAATTTCCCTGTTGGGTTATTGTATTCATGATGAGACAAGGTTCATCATTTATGAACTAATGCAGAACGGATCTTTGGAAAGACAATTACATGGACCTTCTCATGGATCGGCTTTAACTTGGCATATCCGGATGAAAATTGCACTTGATACAGCAAGAGCATTAGAATATCTCCATGAGAATTGCAACCCTCCTGTGATCCACAGAGATCTGAAATCATCCAATATACTTTTGGATTCTAATTTCAAGGCCAAGATTTCAGATTTTGGTCTTGCTGTAATTTCTGGGAGTCAAAACAAGAACAACATTAAGCTTTCAGGCACTCTTGGTTATGTTGCTCCAGAATATCTGTTAGATGGTAAATTGACTGACAAAAGTGATGTCTATGCTTTTGGGGTTATCCTTCTAGAACTCCTAATGGGAAGAAAACCTGTAGAGAAAATGACACGAACTCAGTGTCAATCTATCGTTACATGGGCCATGCCTCAACTCACTGATAGATCAAAGCTACCAAACATTGTTGATCCTGTGATTAAAAACACAATGGATTTGAAGCATTTGTTCCAAGTTGCTGCTGTAGCTGTACTGTGTGTACAACCAGAACCAAGTTACCGGCCATTAATCACAGATGTCCTTCACTCCCTCGTACCCCTTGTTCCTGTCGATCTTGGAGG >SEQIDNO: 85EYQLLEAATDNFSESNILGEGGFGCVYKACFDNNFLAAVKRMDVGGQDAEREFEKEVDLLNRIQHPDIISLLGYCIHDETRFIIYELMQNGSLERQLHGPSHGSALTWHIRMKIALDTARALEYLHENCNPPVIHRDLKSSNILLDSNFKAKISDFGLAVISGSQNKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVILLELLMGRKPVEKMTRTQCQSIVTWAMPQLTDRSKLPNIVDPVIKNTMDLKHLFQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPVDLGG >SEQIDNO: 86TGTGCTCATGATGAGACCAAACTACTTGTTTACGAACTTATGCACAATGGTTCGTTAGAAACTCAATTACACGGTCCTTCTTGTGGATCCAATTTAACATGGCATTGTCGGATGAAAATTGCGCTAGATATAGCGAGAGGATTGGAATATTTACATGAACACTGCAAACCATCTGTGATTCATAGAGATTTGAAGTCATCTAACATCCTTTTGGATTCAAAATTCAATGCCAAGCTTTCGGATTTCGGTCTTGCTGTGATGAACGGTGCCAATACCAAAAACATTAAGCTTTCGGGGACGTTGGGTTACGTAGCTCCCGAGTATCTTTTAAATGGGAAATTGACCGATAAAAGTGACGTCTACGCATTCGGAGTTGTACTTTTAGAGCTTCTACTCAAAAGGCGGCCTGTCGAAAAACTAGCACCATCCGAGTGCCAGTCCATCGTCACTTGGGCTATGCCGCAACTAACAGACAGAACAAAGCTTCCGAGTGTTATAGATCCCGTGATCAGGGACACGATGGATCTTAAACACTTGTATCAAGTGGCGGCTGTGGCTGTGTTGTGTGTTCAACCGGAACCGGGATACCGGCCGTTGATAACCGACGTCTTGCATTCTCTGGTTCCTCTCGTGCCGGTTGAACTCGGAGGGACTCTACGAGTTGCGGAAACAGGTTGCGGCACAGTTGACTTATGA >SEQIDNO: 87CAHDETKLLVYELMHNGSLETQLHGPSCGSNLTWHCRMKIALDIARGLEYLHEHCKPSVIHRDLKSSNILLDSKFNAKLSDFGLAVMNGANTKNIKLSGTLGYVAPEYLLNGKLTDKSDVYAFGVVLLELLLKRRPVEKLAPSECQSIVTWAMPQLTDRTKLPSVIDPVIRDTMDLKHLYQVAAVAVLCVQPEPGYRPLITDVLHSLVPLVPVELGGTLRVAETGCGTVDL >SEQIDNO: 88TGGATTTGGATGCGTTTAAAAGCTCAACTCAATGATAACTTATTAGTTGCGGTCAAACGACTAGACAATAAAAGTCAAAATTCCATCAAAGAATTCCAGACGGAAGTGAATATTTTGAGTAAAATTCAACATCCAAATATAATTAGTTTGTTGGGATATTGCGATCATGATGAAAGCAAGCTACTTGTTTACGAATTGATGCAAAATGGTTCTTTAGAAACTCAGTTACATGGGCCTTCTTGTGGATCCAATTTAACATGGTATTGCCGGATGAAAATTGCCCTAGATATAGCAAGAGGATTGGAATATTTACATGAACACTCCAAACCATCTGTGATTCATAGAGATCTCAAATCATCTAATATACTTCTTGATTCAAATTTCAATGCAAAGCTTTCGGATTTTGGTCTTGCGGTGATGGAAGGTGCAAATAGCAAAAACATTAAACTTTCGGGGACATTGGGATACGTAGCACCCGAATATCTTTTAGATGGGAAATTAACCGATAAAAGTGACGTGTATGCATTTGGAGTCGTACTTTTTGAGCTTTTACTCAGAAGACGACACGTTGAAAAACTAGAATCATCACAATCCCGCCAATCTATTGTCACTTGGGCGATGCCACTACTAATGGACAGATCGAAGCTTCCGAGTGTGATAGATCCTGTGATTAGGGATACAATGGATCTTAAACATCTTTATCAAGTGGCTGCGGTGGCGGTGTTGTGTGTTCAATCGGAACCGAGTTACCGTCCGTTGATAACCGATGTTTTACATTCTCTTGTTCCTCTTGTCCCGGTTGAACTTGGAGGGACACTTAGAGTTGTAGAAAA GAGTGTTGT >SEQIDNO: 89WIWMRLKAQLNDNLLVAVKRLDNKSQNSIKEFQTEVNILSKIQHPNIISLLGYCDHDESKLLVYELMQNGSLETQLHGPSCGSNLTWYCRMKIALDIARGLEYLHEHSKPSVIHRDLKSSNILLDSNFNAKLSDFGLAVMEGANSKNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLFELLLRRRHVEKLESSQSRQSIVTWAMPLLMDRSKLPSVIDPVIRDTMDLKHLYQVAAVAVLCVQSEPSYRPLITDVLHSLVPLVPVELGGTLRVVEKSVV >SEQIDNO: 90ATTCTTTTAGATGCAAACTTCAATGCCAAGCTTTCTGATTTTGGCTTGTCTGTCATTGTTGGAGCACAAAACAAGAATGATATAAAGCTTTCCGGAACGATGGGTTATGTTGCTCCTGAATATCTTTTAGATGGTAAATTGACTGATAAAAGTGATGTCTATGCTTTTGGAGTTGTGCTTTTGGAGCTTCTTTTAGGAAGAAGGCCTGTTGAAAAACTGGCACCATCTCAATGTCAATCCATTGTCACATGGGCTATGCCTCAACTCACTGATAGATCAAAGTTACCCGATATCGTTGATCCGGTGATCAGACACACAATGGACCCTAAACATTTATTTCAGGTTGCTGCTGTCGCCGTGCTGTGTGTGCAACCAGAACCGAGCTATCGTCCCCTAATAACAGATCTTTTGCACTCTCTTATTCCTCTTGTTCCTGTTGAGCTAGGAGGTACTCACAGATCATCAACATCACAAGCTCCTGTGGCTCCAG CTTAG >SEQIDNO: 91ILLDANFNAKLSDFGLSVIVGAQNKNDIKLSGTMGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLAPSQCQSIVTWAMPQLTDRSKLPDIVDPVIRHTMDPKHLFQVAAVAVLCVQPEPSYRPLITDLLHSLIPLVPVELGGTHRSSTSQAPVAPA >SEQIDNO: 92GATGGGAAGCTCACCGAGAAAAGCGACGTGTACGCGTTTGGCATAGTGCTTCTTGAGCTGCTAATGGGAAGGAAGCCTGTTGAGAAGTTGAGTCAATCTCAGTGCCAATCAATTGTGACTTGGGCCATGCCCCAACTGACAGACAGATCAAAACTTCCCAACATAATTGACCCAGTGATCAGGGACACAATGGATCCAAAGCACTTGTATCAGGTTGCAGCAGTGGCTGTTCTATGCGTGCAACCAGAACCGAGTTACAGACCACTGATAACGGATGTTCTCCACTCTTTAGTTCCTCTAGTGCCTGTGGAGCTTGGTGGGACACTAAGGGTTGCAGAGCCACCGTCCCCAAACCAAAATCATTCTCCTCGTTGA >SEQIDNO: 93DGKLTEKSDVYAFGIVLLELLMGRKPVEKLSQSQCQSIVTWAMPQLTDRSKLPNIIDPVIRDTMDPKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRVAEPPSPNQNHSPR >SEQIDNO: 94GGGGTTCATGGCAAGAACAATATAAAACTTTCAGGAACTTTAGGATATGTCGCGCCGGAATACCTTTTAGATGGTAAACTTACTGATAAAAGTGACGTTTATGCGTTTGGAGTTGTGCTTCTCGAGCTTTTGATAGGACGAAAACCCGTGGAGAAAATGTCACCATTTCAATGCCAATTTATCGTTACATGGGCAATGCCTCAGCTAACGGACAGATCGAAGCTTCCTAATCTTGTGGATCCTGTGATTAGAGATACTATGGACTTGAAGCCCTTATATCAAGTTGCGGCTGTAACTGTGTTATGTGTACAACCCGAACCAAGTTACCGCCCATTAATAACGGATGTTTTGCATTCGTTCATCCCACTTGTACCTGCTGATCTTGGAGGGTCGTTAAAAGTTGTCGACTTTTAA >SEQIDNO: 95GVHGKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLIGRKPVEKMSPFQCQFIVTWAMPQLTDRSKLPNLVDPVIRDTMDLKPLYQVAAVTVLCVQPEPSYRPLITDVLHSFIPLVPADLGGSLKVVDF >SEQIDNO: 96ATCGTGTTCCATTTTGGTTGTTGTCTAAAGCTTTCAGATTTTGGTCTTGCTGTAATGGATGGAGCCCAGAACAAAAACAACATCAAGCTTTCAGGGACATTGGGTTATGTAGCTCCAGAGTATCTTTTAGATGGAAAACTGACCGACAAAAGTGATGTATATGCATTTGGAGTTGTACTTTTAGAGCTTCTACTTGGAAGACGGCCTGTAGAAAAACTGGCCGCATCTCAATGCCAATCTATCGTCACTTGGGCCATGCCACAGCTAACAGACAGATCAAAGCTCCCAAATATTGTCGATCCTGTAATCAGATATACGATGGATCTCAAACACTTGTACCAAGTTGCTGCCGTGGCAGTGCTGTGTGTGCAACCAGAGCCAAGTTACCGGCCATTAATAACCGATGTTTTGCATTCTCTTATCCCTCTTGTTCCGGTGGAGCTCGGGGGAACTCTAAAAGCTCCACAAACAAGGTCTTCGGTAACAAATG ACCCGTGA >SEQIDNO: 97IVFHFGCCLKLSDFGLAVMDGAQNKNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLAASQCQSIVTWAMPQLTDRSKLPNIVDPVIRYTMDLKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLIPLVPVELGGTLKAPQTRSSVTNDP >SEQIDNO: 98CGTGGATCAACTTTAAGTTGGCCTCTCCGAATGAAAATTGCTTTGGATATTGCAAGAGGATTAGAATACCTTCACGAGCGTTGCAACCCCCCTGTGATCCATAGGCATCTCAAATCGTCTAATATTCTTCTTGATTCCAGCTTCAACGCAAAGATTTCTGATTTTGGCCTTTCTGTAACTGGCGGAAACCTAAGCAAGAACATAACCAAGATTTCGGGATCACTGGGTTATCTTGCTCCAGAGTATCTCTTAGACGGTAAACTAACTGATAAGAGTGATGTGTATGGTTTTGGCATTATTCTTCTAGAGCTTTTGATGGGTAAAAGGCCAGTGGAGAAAGTGGGAGAAACTAAGTGCCAATCAATAGTTACATGGGCTATGCCCCAGCTTACGGACCGATCAAAGCTTCCGAATATTGTTGACCCTACGATCAGGAACACAATGGATGTTAAGCATTTATATCAGGTTGCGGCTGTAGCTGTGTTATGTGTGCAACCGGAGCCAAGCTATAGGCCATTGATAACTGATGTACTACACTCCTTCATTCCACTTGTACCAAATGAACTCGGGGGGTCGCTTAGGGTAGTGGATTCTACTCCCCATTGCTCATAG >SEQIDNO: 99RGSTLSWPLRMKIALDIARGLEYLHERCNPPVIHRHLKSSNILLDSSFNAKISDFGLSVTGGNLSKNITKISGSLGYLAPEYLLDGKLTDKSDVYGFGIILLELLMGKRPVEKVGETKCQSIVTWAMPQLTDRSKLPNIVDPTIRNTMDVKHLYQVAAVAVLCVQPEPSYRPLITDVLHSFIPLVPNELGGSLRVV DSTPHCS >SEQIDNO: 100TTAGATAATGGCGGACCCGATTGTCAACGAGAATTCGAGAATGAGGTTGATTTGATGAGTAGAATTAGGCATCCAAATGTGGTTTCTTTATTGGGTTATTGCATTCATGGAGAAACCAGGCTTCTTGTCTATGAAATGATGCAAAACGGGACGTTGGAATCGCTATTGCATGGACCATCACATGGATCCTCACTAACTTGGCACATTCGTATGAAGATCGCCCTCGACACAGCAAGAGGCCTCGAGTATCTGCATGAACACTGCGACCCCTCTGTGATCCACCGTGACCTGAAGCCTTCTAACATTCTTTTGGATTCCAACTACAATTCCAAGCTCTCAGACTTTGGTCTTGCAGTCACTGTTGGAAGCCAGAATCAAACCAACATTAAGATTCTAGGGACACTGGGTTACCTTGCACCAGAGTACGTTTTGAATGGCAAATTGACAGAGAAAAGTGATGTGTTTGCTTTTGGAGTTGTCCTGTTGGAGCTTCTCATGGGCAAGAAACCAGTGGAGAAGATGGCATCCCCTCCATGCCAATCCATTGTCACATGGGCGATGCCTCATCTTACTGACAGAATTAAGCTTCCAAATATCATTGATCCTGTTATTAGAAACACCATGGATCTGAAACACTTGTACCAGGTTGCAGCTGTTGCTGTTCTCTGCGTACAACCAGAGCCCCAGTTATCGTCCTCTGATAACTGA >SEQIDNO: 101LDNGGPDCQREFENEVDLMSRIRHPNVVSLLGYCIHGETRLLVYEMMQNGTLESLLHGPSHGSSLTWHIRMKIALDTARGLEYLHEHCDPSVIHRDLKPSNILLDSNYNSKLSDFGLAVTVGSQNQTNIKILGTLGYLAPEYVLNGKLTEKSDVFAFGVVLLELLMGKKPVEKMASPPCQSIVTWAMPHLTDRIKLPNIIDPVIRNTMDLKHLYQVAAVAVLCVQPEPQLSSSDN >SEQIDNO: 102TCGGCTCGGCCCAGAACAAGATCGCAAGAC >SEQIDNO: 103CTACATTCTCTCCTCGTATTATTCCTCGTTGACT >SEQIDNO: 104ACTTTCAGATGAGTGGATCATAACCCTATACA >SEQIDNO: 105AGATACAATGGATCTCAAACACTTATACCAG >SEQIDNO: 106AAAGGATCCATGGGAAGTGGTGAAGAAGATAGATTTGATGCT >SEQIDNO: 107TTTCTGCAGTCTGTGAATCATCTTGTTAACCGGAGAGTCC >SEQIDNO: 108TCTGAGTTTTAATCGAGCCAAGTCGTCTCA >SEQIDNO: 109TATCCCGGGAAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTC AGTC >SEQIDNO: 110TTTGGATCCTGTGAATCATCTTGTTAACCGGAGAGTCC >SEQIDNO: 111ATACCCGGGTCTGTGTCAGGAATCCAAATGGGAAGTGGTGA >SEQIDNO: 112AAAGGATCCTCTGTGTCAGGAATCCAAATGGGAAGTGGTGA >SEQIDNO: 113AAATCTAGACTGTGAATCATCTTGTTAACCGGAGAGTCC >SEQIDNO: 114ATAGAGCTCGCAAGAACCAATCTCCAAAATCCATC >SEQIDNO: 115ATAGAGCTCGAGGGTCTTGATATCGAAAAATTGCACG >SEQIDNO: 116ATAGGATCCTCGCAAGAACCAATCTCCAAAATCCATC >SEQIDNO: 117ATATCTAGACTCGAGGGTCTTGATATCGAAAAATTGCACG >SEQIDNO: 118ATATCTAGAAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCA GTC >SEQIDNO: 119ATAGGATCCTGTTAAAAGCGATTTATAATTTACACCGTTTTGGTGTA >SEQIDNO: 120ATACCCGGGAAAAGTTTTTGATGAAATTCAATCTAAAGACT >SEQIDNO: 121AAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTCAGTCTCTAATTCTTTTGATGATCTTCATCACTGTCTCTGCTTCTTCTGCTTCAAATCCTTCTTTAGCTCCTGTTTACTCTTCCATGGCTACATTCTCTCCTCGAATCCAAATGGGAAGTGGTGAAGAAGATAGATTTGATGCTCATAAGAAACTTCTGATTGGTCTCATAATCAGTTTCTCTTCTCTTGGCCTTATAATCTTGTTCTGTTTTGGCTTTTGGGTTTATCGCAAGAACCAATCTCCAAAATCCATCAACAACTCAGATTCTGAGAGTGGGAATTCATTTTCCTTGTTAATGAGACGACTTGGCTCGATTAAAACTCAGAGAAGAACTTCTATCCAAAAGGGTTACGTGCAATTTTTCGATATCAAGACCCTCGAGAAAGCGACAGGCGGTTTTAAAGAAAGTAGTGTAATCGGACAAGGCGGTTTCGGATGCGTTTACAAGGGTTGTTTGGACAATAACGTTAAAGCAGCGGTCAAGAAGATCGAGAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGACTTGTTGAGCAAGATCCATCACTCGAACGTTATATCATTGTTGGGCTCTGCAAGCGAAATCAACTCGAGTTTCATCGTTTATGAGCTTATGGAGAAAGGATCATTAGATGAACAGTTACATGGGCCTTCTCGTGGATCAGCTCTAACATGGCACATGCGTATGAAGATTGCTCTTGATACAGCTAGAGGACTAGAGTATCTCCATGAGCATTGTCGTCCACCAGTTATCCACAGAGATTTGAAATCTTCGAATATTCTTCTTGATTCTTCCTTCAACGCCAAGATTTCAGATTTCGGTTTTGCTGTATCGCTGGATGAACATGGCAAGAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATACCTCCTTGACGGAAAACTGACGGATAAGAGTGATGTTTATGCATTTGGGGTAGTTCTGCTTGAACTCTTGTTGGGTAGACGACCAGTTGAAAAATTAACTCCAGCTCAATGCCAATCTCTTGTAACTTGGGCAATGCCACAACTTACCGATAGATCCAAGCTTCCAAACATTGTGGATGCCGTTATAAAAGATACAATGGATCTCAAACACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTGCAGCCAGAACCAAGTTACCGGCCGTTGATAACCGATGTTCTTCACTCACTTGTTCCACTGGTTCCGGTAGAGCTAGGAGGGACTCTCCGGTTAACAAGATGATTCACAG >SEQIDNO: 122TCGGACAAGGCGGTTTCGGATGCGT >SEQIDNO: 123TAGTCCTCTAGCTGTATCAAGAGCAATCTTCA >SEQIDNO: 124TATCATTGTTGGGCTCTGCAAGTGAAATCAAC >SEQIDNO: 125TGGAGAAAGGATCCTTAGATGATCAGTTACAT >SEQIDNO: 126TCCATGTAACTGATCATCTAAGGATCCTTTC >SEQIDNO: 127ATAAACGACGAAACTCGAGTTGATTTCACTTGCAGAG >SEQIDNO: 128AAAATGAAGAAACTGGTTCATCTTCAGT >SEQIDNO: 129TAGACTTCTATTCTCACATTCTTACAC >SEQIDNO: 130TCCAATGATCCATTATGCATCAGCTCA >SEQIDNO: 131TCGTTCTCAAATTCTCTCTCAGCATGTTG >SEQIDNO: 132TCCGGATATGCCAGGTCAGCGCTGATCCA >SEQIDNO: 133TCCAGGGATCCCTTCTCCATGAGCTCAT >SEQIDNO: 134AAAGAGCTCTCTGTGTCAGGAATCCAAATGGGAAGTGGTGA >SEQIDNO: 135ATAGCTAGCTGTTAAAAGCGATTTATAATTTACACCGTTTTGGTGTA >SEQIDNO: 136ATAGCTAGCAGAAAAGTTTTTGATGAAATTCAATCTAAAGACT >SEQIDNO: 137TCTGGGTTTATCATCATACCAAGTATCCA >SEQIDNO: 138ATTCAGTTCCATCAAGATTGTTGGCATGGAC >SEQIDNO: 139TGGAGGGAGGTGGCCCTGAGTGCGAGAAGGA >SEQIDNO: 140GCTGGATCTGCTTGGCAGGATTCGGCA >SEQIDNO: 141ATATCTAGATGCTAGGTTATAGATCCATGCA >SEQIDNO: 142ATAGGATCCACCAGAACTATATATACGAAGGCA >SEQIDNO: 143AGGACGACTTGGCTCGATTAAAATCACAGGTCGTGATATG >SEQIDNO: 144TAATCGAGCCAAGTCGTCCTACATATATATTCCTA >SEQIDNO: 145TAATCGAGCCAAGTCGTCCTCTCTTTTGTATTCCA >SEQIDNO: 146AGGACGACTTGGCTCGATTAAAATCAAAGAGAATCAATGATC >SEQIDNO: 147GACGACTTGGCTCGATTAAAA >SEQIDNO: 148TGCTAGGTTATAGATCCATGCAAATATGGAGTAGATGTACAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTGATGTTTTAGGAATATATATGTAGAGAGAGCTTCCTTGAGTCCATTCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATTGGACTGAAGGGAGCTCCCTCTCTCTTTTGTATTCCAATTTTCTTGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAG TTCTGGT >SEQIDNO: 149TGCTAGGTTATAGATCCATGCAAATATGGAGTAGATGTACAAACACACGCTCGGACGCATATTACACATGTTCATACACTTAATACTCGCTGTTTTGAATTGATGTTTTAGGAATATATATGTAGGACGACTTGGCTCGATTAAAATCACAGGTCGTGATATGATTCAATTAGCTTCCGACTCATTCATCCAAATACCGAGTCGCCAAAATTCAAACTAGACTCGTTAAATGAATGAATGATGCGGTAGACAAATTGGATCATTGATTCTCTTTGATTTTAATCGAGCCAAGTCGTCCTCTCTTTTGTATTCCAATTTTCTTGATTAATCTTTCCTGCACAAAAACATGCTTGATCCACTAAGTGACATATATGCTGCCTTCGTATATATAG TTCTGGT >SEQIDNO: 150CTTAGCCAATGGATGAGGATGACACGATAATGATAATCAAAGATCAACATGGCACGCTCAAGACCGCCTTTAGAAGTCCTCTCTAAATTCTTTCTTCCGATCTCCTAAATATGTTTTGTTTTGGTCAAATAAATTGATAGGTAATACTTAGTGATTATACTATTTGGTTTTTGTTTTATCATTGACTATTTCACTTTTATAAATCAAATACTTATCAAAATTGTTCTTTCCGTATGTATTCATATTTTCTAATATTGTAAAGATTTGTTTCACCTAACATCTGTACCCATCTTTGATCATTGACAAAATATATATTAGAATGGCCTTAGAACGTGTTAGGCATCTTCCTACTATTATCATATTACCTAATCCCCAATTTTATTACATTTTTTAATTTCTAAAAGAGCTTGAATATAATGTCATTTCGAATATCTCTGTTCATCTTTTTTTTTTTCTGTGCGACTTCTGACCCAAAGCCTTCGACGATTTTTTCCAATCTGAAAACTTTTGAATAAGGAACTTAGTCAATGGTCAACACCTTGCTAATTAAACAAAGTTCCATTGATACAATAATGAGATTTTTGTACATTAACGCTTTCATATAGTTTTTGCGATTCAACAGATAATCTTAAAATTAAGGAGTCCTATTGATAAAGTCTTGTTCAAACGTACAAACTCAATCCACACAAAACCTTCATAAAATACGATATAGGAAATAAAGATTGTTTTTGCGTGAGAAAATACTATATGAACTCAAAAGATTTTAAAACAATTTGTATTAATACATAAACAATTGTTGTGATACACCCGTGTAAAATTTTAAGATTGTTTTTTTCTGAAATTCTTCAAGGAAACTTATAGCTTAAAATCTACACTTCAAATACTCTGTTTTAAAGGCATTAAAAATAACTGCGTTTCAGAAAAATATTGAAATTTTAGCTGATCTTTTGCTACAAATTTAAGGAATCTTGGCACCTGCAGAATCTATAACATGTTCATTAAGTAATGCAATAGTTATACAATTATACATTATTTGCATCATACTTATATTATAGTGATATTAACAAACCCATGTTCTCAGCACACTTTTACGTAGAAAAACATAAAAACCCAAATAGGAAGAAGCCACTCATAAGGATAATGGGTTTATATAATTCACAGCAAAGAAAGCCATCGAACTATTCGATTAATTATCCATTCTTTTTTTTTTTAGTTTGAATGTATAAGAACAAAGAGTTGTTACGCATCATGACAATGTCTTAGAAAACAAAAGAAATGAATAAAAAAGTAAAACGAAAAATAAAAAGTGAGGATGAAGTTGTTGAATGAGTTGGCGAGGCGGCGACTTTTTCATACATTCCATTTACTTAATTCCTAAAGTCCTTCTCACATCTCTTTGTTATATAATGACACCATAACCATTTCTTCTCTTCACAATCTTTACAAGAATATCTCTCTTCTACAGTAAAC AAAAA >SEQIDNO: 151ACGTAAGCTTCTTAGCCAATGGATGAGGATG >SEQIDNO: 152ACGTTCTAGATTTTTGTTTACTGTAGAAGAG >SEQIDNO: 153TGCTGCTTCAAATCCTTCTATAGCTCCTGTTTATACCACCATGACTACTTTCTCTCCAGGAATTCAAATGGGAAGTGGTGAAGAACACAGATTAGATGCACATAAGAAACTCCTGATTGGTCTTATAATCAGTTCCTCTTCTCTTGGTATCGTAATCTTGATTTGCTTTGGCTTCTGGATGTACTGTCGCAAGAAAGCTCCCAAACCCATCAAGATTCCGGATGCTGAGAGTGGGACTTCATCATTTTCAATGTTTGTGAGGCGGCTAAGCTCAATCAAAACTCAGAGAACATCTAGCAATCAGGGTTATGTGCAGCGTTTCGATTCCAAGACGCTAG >SEQIDNO: 154TATGGATCCTGCTGCTTCAAATCCTTCTATAGCTCCTG >SEQIDNO: 155TATTCTAGACTAGCGTCTTGGAATCGAAACGCTGCAC >SEQIDNO: 156TATGAGCTCTGCTGCTTCAAATCCTTCTATAGCTCCTG >SEQIDNO: 157TATGAGCTCCTAGCGTCTTGGAATCGAAACGCTGCAC >SEQIDNO: 158GCAGATCGCTCCTCCCGTCGTGAT >SEQIDNO: 159CGCCTAGGAGCGACGGGTACTCGATCAT >SEQIDNO: 160CCTAGCTAAGCGACGGGTACTCGATCAT >SEQIDNO: 161GCTCCTCCCGTCGTGATCACAGTGGTGAGGCACCACCATTACCACCGGGAGCTGGTCATCTCCGCTGTCCTCGCCTGCGTCGCCACCGCCATGATCCTCCTCTCCACACTCTACGCCTGGACGATGTGGCGGCGGTCTCGCCGGACCCCCCACGGCGGCAAGGGCCGCGGCCGGAGATCAGGGATCACACTGGTGCCAATCCTGAGCAAGTTCAATTCAGTGAAGATGAGCAGGAAGGGGGGCCTTGTGACGATGATCGAGTACCCGTCGCT >SEQIDNO: 162CGGGATCCCGGCATAACAAACTCGTGCATCC >SEQIDNO: 163CCATCGATGGCGCCAAACACAATAGCTCAA >SEQIDNO: 164GTAAGTAATTTCAAGTTTAAGTTTCATAAGCATAACAAACTCGTGCATCCAATTTGAACCATTTTACTGTCCTGGCATCCTCTAAATATTTCCTTGATTATCAGCTTATCTTCATCCCATTGAATCAGAAAATTACCAACCCTTGTTTTAGCTTTAATCATTGTTATTTGTTGTCTGAGGGGCTACACTGTTTCTTTATATTGGTGAAGGAGTTACCAGGCAAAAATTCCCACCTCCTGATATTAGCAGAGACCCCCTTTTTTGTGCCTGTATGCATACTAACAAATAATACAGATGGAAATATGTATATTTGTTATATCATGGATTGATGCTTTATGTTTAGCAAGTCCATGCAATGGTAGTCAAAAGATGTAAACTTTTGAATGATATATTGGGGCTTTAGATTAGCCATTTTTACCCTCACTTGAAAATGACAATTTTGCCCTTCCGATCTACTTTCTCTTGTCACCTCAGGCAGGCTCTTGAAAGTTCTTATCCCTGAATTCCGTGGAAGTTTATTATTCTAATGTTATAGTTTACTTAAAGTGTCGCATAATCTACTAGAGCCTAATGGAAGTACTGATGGACTTTGTTTTGCTACAATCACTGCTTGCAAGAATGACTACTTTGGGGCATTTCTAATATATTATTGATATTTCTATGATGTATTGTTGTCCATGTACTTCAGTCCTTACAGCGACTAGTCCTATTTCTGCATTGATAAATTGTTCACTGTCAGACCATCTTGAGTGGCAAGAATGAGTATAACATGTCTTGTTTTTCTGTGATTTCAAGGTAAGCGCACATGCGCACAGTGTACACCGTCACCACATGTGAGTACACCCCCTAGTACACATGTAAAAAAAGCACAGTCCAGTTATTAAATGGACCATTGGCATTGATTGTCGTGTTTATAGGAGTAAAGATACATGTAAACACTAATTCATTGGGAGATATAAATTTATACTACCATTGAATGTGACATAGGCTCTAAGGTTTTTAGTTCAGCATTTCGAAAGAGCTTTGTTTGGTTGGCTTGGGATGGAATCAGGTGACAACATTTTTGGGTTGCAGCAAATTTAATATTGATTGAGGAGGCATACAACGAAATCATTGAGCTATTGTGTTTGGCGTTACATCTATGGAATTTCTTCTAATCTGATTATTGTTTGTA >SEQIDNO: 165GATCCGCTCCTCCCGTCGTGAT >SEQIDNO: 166AACGCGATCGCTTGCATGCCTGCAGTAGAC >SEQIDNO: 167GACTTAATTAAGAATTCGAGCTCGGGTA >SEQIDNO: 168TCGTAGTGCACCACCATTTCCACCGCGAGCTGGTCATCGCCGCCGTCCTCGCCTGCATCGCCACCGTCACGATCTTCCTTTCCACGCTCTACGCTTGGACACTATGGCGGCGATCTCGCCGGAGCACCGGCGGCAAGGTCACCAGGAGCTCAGACGCAGCGAAGGGGATCAAGCTGGTGCCGATCTTGAGCAGGTTCAACTCGGTGAAGATGAGCAGGAAGAGGCTGGTTGGGATGTTCGAGTAC CCGTCG >SEQIDNO: 169GCAGATCTCGTAGTGCACCACCATTTC >SEQIDNO: 170CGCCTAGGCGACGGGTACTCGAACATC >SEQIDNO: 171CCTAGCTACGACGGGTACTCGAACATC >SEQIDNO: 172GATCCTCGTAGTGCACCACCATTTC >SEQIDNO: 173CTCGTAGTGCACCACCATTTC >SEQIDNO: 174AATGGGACCGCCTCCGTTGCTCCGGCGGTGCCGGCGCCGCCTCCCGTCGTGATCATCGTGGAGCGGCGCCATCATTTCCACCGCGAGCTAGTCATCGCCTCCGTTCTCGCCTCCATCGCCATCGTCGCGATTATCCTCTCCACGCTCTATGCGTGGATCCTGTGGCGGCGGTCTCGCCGGCTGCCCAGCGGCAAGGGCGCCAGGAGCGCAGACACCGCGAGGGGAATCATGCTGGTGCCGATCCTGAGCAAGTTCCACTCA >SEQIDNO: 175 GCAGATCAATGGGACCGCCTCCGTTG >SEQIDNO: 176CGCCTAGGTGAGTGGAACTTGCTCAGGA >SEQIDNO: 177CCTAGCTATGAGTGGAACTTGCTCAGGA >SEQIDNO: 178GTAAGTATTCTTGCAACACATTACTATTTTCAATAACCACAAGTTTAAAAGCTTGAGTCCATTTCGCAAACCAGTTGTTCATAACCAAATTCTTAGGTAATTAGGTCCAATTGAGAAAATCTGATCATTGAACACTAGCAGGAAATAACTCAGACATAGTTTCTGCATACTATAATGATGCTTAATATATTTGTTCTCTTTTGAGATTGTATTGCATAGACATTTCTGTGTAAAATAATGTTTTACATCATGTATATATATCACTTTTTATAG >SEQIDNO: 179CGGGATCCTTCTTGCAACACATTACTATTT >SEQIDNO: 180CCATCGATGAAATGTCTATGCAATACAATCTCAA >SEQIDNO: 181GATCCAATGGGACCGCCTCCGTTG >SEQIDNO: 182CAATGGGACCGCCTCCGTTGA >SEQIDNO: 183GGCCCCGGCCGCGCGCGTCTCCGTGTCCTCCGCGACTGTGCACGTTTCGTCGGGAGCGGCGTGCCCACGCCCACCCCCCGTCCACCAGCCAGCAACCGACGGCACTGGTGACACGCGGCTGGTCCGCTCGGTCCGCCCCGCGGCTCCAGATCACGGCAAGCGCGCCCGCCGCCCGCTGCTGCGCTGCGCTGCACGTCCCGCCCTGACGCCACGCCACGCCAAGCGCGACACGACACGACACGACACGACCCGACCCCCGCCAACGAAACGCCGAAACGCGGCAACGCGTGACGGGCGCGCATGGTCGATGCTCTACCCGCGCGTCCGCCCCACGCCAATCTCCCGGCGGGTCCCTCGTGGGACGGGGAACGCGATGCGGCTGCAGGCTGCGACCGCGACCGCGACCGCGACCGCGCCCACGTGAAGGCAGGCAGGCAGCCCCGGAGCGGGCGCGGCGGTGGGCCAACGACGCGTTGCCGTCGCGAATCTTCTTCTGGCCACGGCCAAGGGCCAATCGCCCGCTCCGCTCCGCTCCGCACTCCGCCTCCGCTAGGGAATATGGAACCCGATCCCACGGCCCTCTGGGTCTGGTCGACGGGTCCTCTCGCCGTGGCAGCTGCTTCCCGGACCGGAGGATCGCTGAGCGCGGACGCCACTGCCATTGCCGTCCGACTATAGTTGTTAATTACCATAAAATAATTTGTTAACGATAAAACCCGTGTCAGGCACCGTCGTCTGGACGCTGCTATGGGATAACCATTCGCGTACGTCGGTTGTATGGGTGGGATCCTCTGCGGCACGCCATTCTGGTGCTGCTAGTGGAATAGACAAAAAAAGGGCCGACGGTGTTTGCTCGTGGCAGGCCACACAGAGTGACAACCAGAGTGGTTGCCGCAAAAACAACCAATCACACAAAAAGTGTTGTACCGGTGGAGGACAGCCATTAATCAGCAGGCCGGCTTCGCGGCCAAAAGAAACGGAGAAGAGGAAAAAGGGGGGC >SEQIDNO: 184TCCCAAGCTTGCGCGTCTCCGTGTCCTC >SEQIDNO: 185AGTAAAGCTTCCCCCTTTTTCCTCTTCTCC >SEQIDNO: 186TAATGGTCGAGTGAGGCCCGTATAGATGTAGTTAAATAGCTAAAATTTTTGGAGAAATAAGCATTTTTTTGGAAGAATATATTTAAACATGGGCTTGTAAAACTTGGCTGTAAAGATTTGGAATTTAGGATCTTGGAGCCCCAAAACTGTATAAACTTGCTTAGGGACCCGTGTCTTGTGTGTTGCAGACCAAAAAATTTAGAAAGCATCTAAACACCTATTTGAATGTAAAGTTTACAGCCAAAAGTTTTAGGATGTAAAGATTTGGGATCTAAAAGTAGTCATTAGGAAATAACACGTTAGAGAGAGAGAGTAGATCTTCTTATTGGTTTCTCATGCACTAATCGAACCAATCACTGGACCACTTGAACCAAACTTTATCACATTGAACTTTGTCAGTTCAGTTCGAACGCAGGACTGGAGCTGCCCTTAAGGCCAATTGCTCAAGATTCATTCAACAATTGAAACATCTCCCATGATTAAATCAGTATAAGGTTGCTATGGTCTTGCTTGACAAAGTTTTTTTTTTGAGGGAATTTCAACTAAATTTTTGAGTGAAACTATCAAATACTGATTTTAAAAATTTTTTATAAAAGGAAGCGCAGAGATAAAAGGCCATCTATGCTACAAAAGTACCCAAAAATGTAATCCTAAAGTATGAATTGCATTTTTTTTGTTTGGACGAAAGGAAAGGAGTATTACCACAAGAATGATATCATCTTCATATTTAGATCTTTTTTGGGTAAAGCTTGAGATTCTCTAAATATAGAGAAATCAGAAGAAAAAAAAACCGTGTTTTGGTGGTTTTGATTTCTAGCCTCCACAATAACTTTGACGGCGTCGACAAGTCTAACGGACACCAAGCAGCGAACCACCAGCGCCGAGCCAAGCGAAGCAGACGGCCGAGACGTTGACACCTTCGGCGCGGCATCTCTCGAGAGTTCCGCTCCGGCGCTCCACCTCCACCGCTGGCGGTTTCTTATTCCGTTCCGTTCCGCCT >SEQIDNO: 187AACTGCAGGGTCGAGTGAGGCCCGTA >SEQIDNO: 188TTCTGCAGGGAACGGAACGGAATAAGAA >SEQIDNO: 189GCCGTGGGTCGTTTAAGCTGCCGCTGTACCTGTGTCGTCTGGTGCCTTCTGGTGTACCTGGGAGGTTGTCGTCTATCAAGTATCTGTGGTTGGTGTCATGAGTCAGTGAGTCCCAATACTGTTCGTGTCCTGTGTGCATTATACCCAAAACTGTTATGGGCAAATCATGAATAAGCTTGATGTTCGAACTTAAAAGTCTCTGCTCAATATGGTATTATGGTTGTTTTTGTTCGTCTCCT >SEQIDNO: 190TAGGTACCGCCGTGGGTCGTTTAAGCT >SEQIDNO: 191AAGGTACCAGGAGACGAACAAAAACAA >SEQIDNO: 192AACGCGATCGTAATGGTCGAGTGAGGCCCGTATA >SEQIDNO: 193ATGAAGAAACTGGTTCATCTTCAGTTTCTGTTTCTTGTCAAGATCTTTGCTACTCAATTCCTCACTCCTTCTTCATCATCTTTTGCTGCTTCAAATCCTTCTATAGCTCCTGTTTATACCACCATGACTACTTTCTCTCCAGGAATTCAAATGGGAAGTGGTGAAGAACACAGATTAGATGCACATAAGAAACTCCTGATTGGTCTTATAATCAGTTCCTCTTCTCTTGGTATCGTAATCTTGATTTGCTTTGGCTTCTGGATGTACTGTCGCAAGAAAGCTCCCAAACCCATCAAGATTCCGGATGCTGAGAGTGGGACTTCATCATTTTCAATGTTTGTGAGGCGGCTAAGCTCAATCAAAACTCAGAGAACATCTAGCAATCAGGGTTATGTGCAGCGTTTCGATTCCAAGACGCTAGAGAAAGCGACAGGCGGTTTCAAAGACAGTAATGTAATCGGACAGGGCGGTTTCGGATGCGTTTACAAGGCTTCTTTGGACAGCAACACTAAAGCAGCGGTTAAAAAGATCGAAAACGTTAGCCAAGAAGCAAAACGAGAATTTCAGAATGAAGTTGAGCTGTTGAGCAAGATCCAGCACTCCAATATTATATCATTGTTGGGCTCTGCAAGTGAAATCAACTCGAGTTTCGTCGTTTATGAGTTGATGGAGAAAGGATCCTTAGATGATCAGTTACATGGACCTTCGTGTGGATCCGCTCTAACATGGCATATGCGTATGAAGATTGCTCTAGATACAGCTAGAGGATTAGAGTATCTCCATGAACATTGTCGTCCACCAGTTATCCACAGGGACCTGAAATCGTCTAATATACTTCTTGATTCTTCCTTCAATGCCAAGATTTCAGATTTTGGTCTGGCTGTATCGGTTGGAGTGCATGGGAGTAACAACATTAAACTCTCTGGGACACTTGGTTATGTTGCCCCGGAATATCTCCTAGACGGAAAGTTGACGGATAAGAGTGATGTCTATGCATTTGGGGTGGTTCTTCTTGAACTTTTGTTGGGTAGAAGGCCGGTTGAGAAATTGAGTCCATCTCAGTGTCAATCTCTTGTGACTTGGGCAATGCCACAACTTACCGATAGATCGAAACTCCCAAACATCGTGGATCCGGTTATAAAAGATACAATGGATCTTAAGCACTTATACCAGGTAGCAGCCATGGCTGTGTTGTGCGTTCAGCCAGAACCGAGTTACCGGCCGCTGATAACCGATGTTCTTCACTCACTTGTTCCATTGGTTCCGGTCGAACTAGGAGGGACTCTCCGGTTAACCCGATGA >SEQIDNO: 194MKKLVHLQFLFLVKIFATQFLTPSSSSFAASNPSIAPVYTTMTTFSPGIQMGSGEEHRLDAHKKLLIGLIISSSSLGIVILICFGFWMYCRKKAPKPIKIPDAESGTSSFSAVVRRLSSIKTQRTSSNQGYVQRFDSKTLEKATGGFKDSNVIGQGGFGCVYKASLDSNTKAAVKKIENVSQEAKREFQNEVELLSKIQHSNITSLLGSASEINSSFVVYELMEKGSLDDQLHGPSCGSALTWHMRMKIALDTARGLEYLHEHCRPPVIHRDLKSSNILLDSSFNAKISDFGLAVSVGVHGSNNIKLSGTLGYVAPEYLLDGKLTDKSDVYAFGVVLLELLLGRRPVEKLSPSQCQSLVTWAMPQLTDRSKLPNIVDPVIKDTMDLKHLYQVAAMAVLCVQPEPSYRPLITDVLHSLVPLVPVELGGTLRLTR >SEQIDNO: 195AATCCAGCTCATTCTGGAATTCCTTCTCGCA >SEQIDNO: 196TGAACTTGCTCAGGATTGGCACCAGTGTGATC >SEQIDNO: 197MEIPAAPPPPLPVLCSYVVFLLLLSSCSLARGRIAVSSPGPSPVAAAVTANETASSSSSPVFPAAPPVVITVVRHHHYHRELVISAVLACVATAMILLSTLYAWTMWRRSRRTPHGGKGRGRRSGITLVPILSKFNSVKMSRKGGLVTMIEYPSLEAATGKFGESNVLGVGGFGCVYKAAFDGGATAAVKRLEGGGPDCEKEFENELDLLGRIRHPNIVSLLGFCVHGGNHYIVYELMEKGSLETQLHGSSHGSALSWHVRMKIALDTARGLEYLHEHCNPPVIHRDLKPSNILLDSDFNAKIADFGLAVTGGNLNKGNLKLSGTLGYVAPEYLLDGKLTEKSDVYAFGVVLLELLMGRKPVEKMSPSQCQSIVSWAMPQLTDRSKLPNIIDLVIKDTMDPKHLYQVAAVAVLCVQPEPSYRPLITDVLHSLVPLVPAELGGTLRVAEPPSPSPDQRHYPC >SEQIDNO: 198TATACCGGTAAAATGAGAGAGCTTCTTCTTCTTCTTCTTCTTCATTTTC AGTC >SEQIDNO: 199ATATACCGGTCTTGTTAACCGGAGAGTCCCTCCTAGCTC >SEQIDNO: 200 CGCTCCTCCCGTCGTGAT

LITERATURE

-   1. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. (1990)    Basic local alignment search tool. J. Mol. Biol. 215: 403-410.-   2. Altschul S F, Madden T L, Schäffer A A, Zhang J, Zhang Z, Miller    W, Lipman D J. (1997) Gapped BLAST and PSI-BLAST: a new generation    of protein database search programs. Nucl. Acid Res. 25: 3389-3402.-   3. An G, Mitra A, Choi H K, Costa M A, An K, Thornburg R W, Ryan    C A. (1989) Functional analysis of the 3′ control region of the    potato wound-inducible proteinase inhibitor II gene. Plant Cell 1:    115-122.-   4. Araus L J, Slafer G A, Reynolds M P, Royo C (2002) Plant Breeding    and drought in C3 cereals: What should we breed for? Annals of    Botany 89: 925-940.-   5. Atanassvoa R, Chaubet N, Gigot C (1992) A 126 bp fragment of a    plant histone gene promoter confers preferential expression in    meristems of transgenic Arabidopsis. Plant Journal 2(3): 291-300.-   6. Beetham P R, Kipp P B, Sawycky X L, Arntzen C J, May G D (1999) A    tool for functional plant genomics: chimeric RNA/DNA    oligonucleotides cause in vivo gene-specific mutations. Proceedings    of the National Academy of Science USA, 96: 8774-8778.-   7. Bevan M, Barnes W M, Chilton M D (1983) Structure and    transcription of the nopaline synthase gene region of T-DNA. Nucl.    Acids Res. 12: 369-385.-   8. Bevan M, Barker R, Goldsbrough A, Jarvis M, Kavanagh T,    Iturriaga G. (1986) The structure and transcription start site of a    major potato tuber protein gene. Nucleic Acids Research 14:    4625-4636.-   9. Christensen A H, Sharrock R A, Quail P H. (1992) Maize    polyubiquitin genes: structure, thermal perturbation of expression    and transcript splicing, and promoter activity following transfer to    protoplasts by electroporation. Plant Mol. Biol. 18: 675-689.-   10. 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1. A method of increasing water use efficiency in a plant, comprising:a) transforming a plant, a plant tissue culture or a plant cell with avector comprising a nucleic acid construct that inhibits the expressionor activity of a PK220 gene to obtain a transformed plant, a transformedplant tissue culture or a transformed plant cell; b) growing thetransformed plant or regenerating a plant from the transformed planttissue culture or the transformed plant cell; and c) selecting a planthaving increased water use efficiency relative to a wild type plant. 2.The method of claim 1, wherein the nucleic acid construct comprises anantisense sequence of a nucleic acid sequence encoding a PK220polypeptide.
 3. The method of claim 2, wherein the antisense sequence isof at least 21 nucleotides.
 4. The method of claim 2, wherein thenucleic acid sequence is selected from the group consisting of SEQ IDNOs: 24, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 84, 86, 88, 90, 92,94, 96, 98, 100, 153, 161 and
 193. 5. The method of claim 1, wherein thenucleic acid construct encodes a mutant PK220 polypeptide that lacksfull functionality of the wild type PK220 polypeptide.
 6. The method ofclaim 5, wherein the nucleic acid construct comprises a nucleic acidsequence of SEQ ID NO: 3 or
 5. 7. The method of claim 1, wherein thenucleic acid construct comprises a constitutive promoter, an induciblepromoter or a tissue specific promoter.
 8. The method of claim 7,wherein the tissue specific promoter is a root promoter.
 9. A transgenicplant produced by the method of claim 1, wherein the transgenic planthas increased water use efficiency relative to a wild type plant.
 10. Atransgenic seed produced by the transgenic plant of claim 9, wherein thetransgenic seed produces a plant having increased water use efficiencyrelative to a wild type plant.